Document QXjYqVO5M0Z649K5ELZjeaE6o

EPA 560/6-81-002 9 EPIDEMIOLOGY STUDIES SCREENING FOR THE EARLY DETECTION OF DISEASE IN INDIVIDUALS EXPOSED TO VINYL CHLORIDE JANUARY 1981 FINAL REPORT U.S. ENVIRONMENTAL PROTECTION AGENCY OFFICE OF PESTICIDES AND TOXIC SUBSTANCES WASHINGTON, D.C. CMA 003688 EPA 560/6-81-002 January 1981 SCREENING FOR THE EARLY DETECTION OF DISEASE IN INDIVIDUALS EXPOSED TO VINYL CHLORIDE Carlos H. Tamburro1 Charles Kupchella1 Kenneth Taylor Emanuel Landau^ by Richard Greenberg1 ? Hildegarde Maricq Joseph Whelan, Jr.1 c Joseph Seifter 1. University of Louisville Louisville, Kentucky 2. University of South Carolina Charleston, South Carolina 3. Yale University New Haven, Connecticut 4. American Public Health Association Washington, D. C. 5. U.S. Environmental Protection Agency Washington, D. C. Project Officer Jane Keller Office of Pesticides and Toxic Substances Washington, D.C. U.S. ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460 CMA 003689 DISCLAIMER This project has been funded with Federal Funds from the Environmental Protection Agency under contract number 68-01-3859. The content of this publication does not necessarilyref1ect the views or policies of the U.S. Environmental Protection Agency, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. CMA 003690 DISCLAIMER This project has been funded with Federal Funds from the Environmental Protection Agency under contract number 68-01-3859. The content of this publication does not necessarilyref1ect the views or policies of the U.S. Environmental Protection Agency, nor does mention of trade names, commercial products, or organizations imply a" tor c omer.t by the '3.5. Government. CMA 003691 ABSTRACT A prospective collaborative study was conducted to compare the effectiveness of four clinical techniques in the detection of liver damage due to vinyl chloride monomer exposure. A chemically exposed and medically monitored worker population was identified by histopathological and biochemical documentation. Three techniques were non-invasive: a) grey scale ultrasonography of the liver, b) microvascular skin capillary assessment, and c) urinary analysis of glycosaminog!yean excretion. The fourth technique was the standard 99mTc sulfur colloid radionucleotide liver spleen scan. The screening studies were performed on a randomly selected single cohort of chemical workers; some of whom were known to have disease. All four techniques were analyzed for their sensitivity and specificity as compared to results of the liver biopsy and biochemical blood test classification. Although all four screening techniques had a sensitivity and specificity sum greater than one, none were significantly better than could be explained by chance or the use of a biased coin. Reclassification of the population into those with more severe biochemical abnormalities improved the sensitivity of all screening tests, but only the sensitivity and specificity sum for the GAG test were statistically significant at the 0.05 level. There was no significant correlation between any pair of screening tests. None of the four screening tests agreed with the biopsy results better than might be obtained by biased coin or chance. These screening studies as presently constituted, do not provide sufficient sensitivity and specificity to warrant their use in conmunity screening for subclinical asymptomatic hepatic injury due-to chemical exposure. CMA 003692 INTRODUCTION 1 The initial reports of primary liver cancers (angiosarcoma) in rats exposed to vinyl chloride by Maltoni, et al (1) and the discovery of similar liver tumors in vinyl chloride polymerization workers by Johnson and Creech (2) has lead to considerable environmental concern regarding comnunities surrounding chemical industries which utilize potentially carcinogenic agents such as vinyl chloride. Several investigators have reported on various screening techniques as effective indicators of vinyl chloride chemical injury. Four such techniques - ultrasono graphy (3), radionucleotide scanning (4), na'ilbed capillary visualization (5), and glycosaminoglycan (GAG) (6) excretion - were reported to have some possible usefulness in detecting early chemical injury to the liver. In order to determine the useability of these techniques for community screening, the American Public Health Association (APHA) and Environmental Protection Agency (EPA) funded a multi-center collaborative study designed to determine the comparative sensitivity and specificity of these various techniques in detecting and identifying chemical related hepatic injury in asymptomatic individuals. Materials and Methods Population Selection The chemical worker population consisted of 1,178 active (Group B, Figure 1) employees as of September 1, 1977, and 70 employees who had had liver biopsies regardless of current employment status (Group A, Figure 1); they were undergoing annual medical screening for the identification of work-related disorders. The medical screening consisted of an annual or semi-annual (for those employees with 10 or more years of employment) comprehensive history and physical examinations, laboratory screening studies consisting of 35 biochemical tests, chest and abdomi nal X-rays, and radionucleotide liver-spleen scan. CMA 003693 The initial reports of primary liver cancers (angiosarcoma) in rats exposed to vinyl chloride by Maltoni, et al (1) and the discovery of similar liver tumors in vinyl chloride polymerization workers by Johnson and Creech (2) has lead to considerable environmental concern regarding communities surrounding chemical industries which utilize potentially carcinogenic agents such as vinyl chloride. Several investigators have reported on various screening techniques as effective indicators of vinyl chloride chemical injury. Four such techniques - ultrasono graphy (3), radionucleotide scanning (4), nailbed capillary visualization (5), and glycosamincglycan (GAG) (6) excretion - were reported to have seme possible usefulness in detecting early chemical injury to the liver. In order to determine the useability of these techniques for cormuinity screening, the American Public Health Association (APHA) and Environmental Protection Agency (EPA) funded a multi-center collaborative study designed to determine the comparative sensitivity and specificity of these various techniques in detecting and identifying chemical related hepatic injury in asymptomatic individuals. Materials and Methods Population Selection The chemical worker population consisted of 1,178 active (Group B, Figure 1) employees as of September 1, 1977, and 70 employees who had had liver biopsies regardless of current employment status (Group A, Figure 1); they were undergoing annual medical screening for the idertification of work-related disorders. The medical screening consisted of an annual or semi-annual (for those employees with 10 or more years of employment) comprehensive history and physical examinations, laboratory screening studies consisting of 35 biochemical tests, chest and abdomi nal X-rays, and radionucleotide liver-spleen scan. CMA 003694 2 This population was selected for the collaborative study to determine the comparative effectiveness of four screening techniques in detecting liver damage as indicated by 1) past histopathological documentation of liver injury of 2) current hepatic dysfunction identified biochemically. Three of the four techniques were included as potential non-invasive pro cedures suitable for determining the effects of vinyl chloride in a comnunity population. These included 1) grey scale ultrasonography of the liver as devel oped by Taylor and colleagues (7, 13, 14), 2) a nailbed skin capillary evaluation of the middle and distal phalanges of the fingers as developed by Maricq and associates (8), and 3) urinary analysis of glycosaminoglycan excretions (GAG) as published by Kupchella and associates (9). The fourth method included for comparison purposes was the standard radio- qq nucleotide liver-spleen scan utilizying mTc colloid and interpreted by Whelan and associates (10). The non-invasive screening studies were performed during a single week on a group randomly selected from all chemical company employees. The workers were selected on the basis of complete medical and work data for 1976-1977, and all those employees who had investigative liver biopsies performed during the screening program (1974-1977). The selection process for these workers is illustrated in Figure 1. The biochemical data and radioisotopic scans were part of the routine medical surveillance system for the employees. The pathological data was based on the last or most recent liver biopsy (s) which were performed for medical reasons, both related and not related to their Work. Positive and negative results were determined as defined in Table,1 which list the technique, the evaluation or evaluators, and the criteria used. The employees targeted for examination were selected by simple random sampling from all available employees. CMA 003695 3 One hundred and twenty one of the targeted 170 employees (71 percent) participate. Twenty six declined to participate or could not be scheduled; 13 did not keep their scheduled appointment. After participation, nine employees were discovered to have been misclassified as biochemically abnormal. They had some biochemical y abnormalities but not all (classified intermediate) and have been eliminated from the final analysis. Four biochemically abnormal individuals had abnormally low test values and they were included in the analysis. Liver Biopsies Liver biopsies were performed by the transjugular technique (11) and pro vided two to five biopsies from various areas of the liver. In addition, some individuals had second biopsies performed by the percutaneous or wedge biopsy via mini-laparotomy procedures. Pathological data was recorded in a computerized format identifying all histological abnormalities in a semi-quantitative fashion. Biopsies were read without knowledge of the individual's medical history or chemical exposure by two pathologists and a hepatologist with extensive experience in hepatic chemical injury. All biopsies were classified as 1) normal, 2) abnormal, a) chemical injury, and b) non-chemical injury. The non-biopsy groups were drawn from those currently employed, and based on biochemical liver "function tests" individuals were sorted into positive, negative and indeterminate for hepatic disease. Only the positive and negative are included in this study. The ultrasonic evaluation and its relative ability to identify hepatic damage due to vinyl chloride has been published elsewhere (3). Microvascular techniques and the method of evaluation by Dr. Maricq and co-workers are also published in part (8). The experimental work on GAG excretion in vinyl chloride workers and the techniques for differentiating the electrophoretic patterns in patients with CMa 003696 . n z re d an. .er.ty crs --2 ta-zazed 170 e-zlcyees zercant) ca-v' ::za'ec s'a zaz.'.e; :o zaz::.;: : - : z a- :c"abj;ad; 13 did not keeo their scheduled appointment. After participation, nine employees were discovered to have been misclassified as biochemically abnormal. They had some biochemical abnormalities but not all (classified intermediate) a^d have been eliminated from the final analysis. Four biochemically abnormal individuals had abnormally low test values and they were included in the analysis. Liver 3ioosies Liver biopsies were performed by the transjugular technique (11) and pro vided two to five biopsies from various areas of the liver. In addition, seme individuals had second biopsies performed by the percutaneous or wedge biopsy via mini-laparotomy procedures. Pathological data was recorded in a computerized format identifying all histological abnormalities in a semi-quantitative fashion. Biopsies were read without knowledge of the individual's medical history or chemical exposure by two pathologists and a hepatologist with extensive experience in hepatic chemical injury. All biopsies were classified as 1) normal, 2) abnormal, a) chemical injury, and b) non-chemical injury. The non-biopsy groups were drawn from those currently employed, and based on biochemical liver "function tests" individuals were sorted into positive, negative and indeterminate for hepatic disease. Only the positive and negative are included in this study. The ultrasonic evaluation and its relative ability to identify hepatic damage due to vinyl chloride has been published elsewhere (3). Microvascular techniques and the method of evaluation by Dr. Maricq and co-workers are also published in part (8). The experimental work on GAG excretion in vinyl chloride workers and the techniques for differentiating the electrophoretic patterns in patients with CMA 003697 4 angiosarcoma and connective tissue damage of the liver has been published else where (9). The effectiveness of radioisotopic (radionucleotide) scanning as a technique for identifying anatomical lesions in vinyl chloride workers is in preparation (12). Method of Analysis The biopsied group and the non-biopsied (biochemical) group were analyzed independently. For the biopsied group sensitivity and specificity were estimated for each screening test by assuming that the biopsy was correct. For the bio chemical group the biochemical classification was assumed to be correct. For each analysis the data consisted of a simple cross classification. In a perfect screening test, the sum of sensitivity and specificity would be two. In a screening test which provided results no better than could be obtained by using a biased coin, the sum of sensitivity and specificity would be equal to one. We, therefore, estimated 95% confidence limits for the sum of sensitivity and spec ificity and observed whether or not one is included within these limits. As a test of statistical significance this is equivalent to the usual X test for independent proportions. Finally, in Table 4 we looked at the association (as measured by the phi coefficient) between each pair of screening tests. For these comparisons we used all employees who received both screening tests regardless of their biopsy status. In this case, we assumed both tests were subject to error and estimated the phi coefficient (r$) between them. Finally, for completeness, we give the biochemical classification for the 51 employees included tn the biopsy group. Results Table 2 compares the histological and biochemical results for the 51 biopsied employees included in the study. Twenty-two of these employees had biochemical abnormalities as defined in Table 1. There was no significant CMA 003698 5 correlation between the biochemical and biopsy classification for the 29 employees with positive or negative biochemical classifications (r$ 3 0.21; xf = 0.20). The biochemical studies used in this analysis were those determined at the time of this and not at the time the biopsy was.performed. In all instances of disagreement, the biopsy was positive and the biochemical results negative (P< 0.001). Figure 2 provides the sensitivity and specificity for each of the screening tests when compared to biopsy results. In no case is the sum significantly greater than one, indicating that the results are not statistically significantly better than could be obtained using the biased coin. With the exception of the GAG studies, similar results are obtained when comparing the sum of sensitivity and specificity in the biochemical group (Figure 3). The sum of sensitivity and specificity for the GAG studies are just statistically significant with 95 percen confidence limits of 1.06 to 1.52. Table 3 gives the frequency distribution of the results of the GAG test for employees with normal and abnormal biochemical results. The distributions differ in their spread (variance) and not in their location (means). Finally, the correlation matrix for the four tests are given in Table 4. There is no significant correlation, as measured by r$, between any pair of the screening tests. This is also true when they are sorted by biopsy status. After reviewing the results, a reclassification of the 51 employees who had biopsies was assessed in regard to whether there was chemically induced liver damage. Ultrasonographic evaluation was reclassified by Dr. Taylor and the liver biopsies by Drs. Tamburro and Popper. Table 5 gives the results of this additional analysis which demonstrates that there was no agreement that could not be explained by chance (P = 0.60). CMA 003699 ' a ' po ^ ~~ ' "5 , *"' " '. ~ .1 ' " ' ) ii " ' ^ " ~ L T* ~ ; ;-r'T./i3c m ' * n S ' b ' `a. C " F' t " ~ *. * ' ' 5 ' ' ~ * il 11 3 3 S 1 T ' C 2 ~ ' v ", " = ,~ ~ 0.20). The biochemical studies used in this analysis were those determined at the time of this and not at the time the biopsy was performed. In all instances of disagreement, the biopsy was positive and the biochemical results negative (P < 0.001). Figure 2 provides the sensitivity and specificity for each of the screening tests when ccmpared to biopsy results. In no case is the sum significantly greater than one, indicating that the results are not statistically significantly better than could be obtained using the biased coin. With the exception of the GAG studies, similar results are obtained when comparing the sum of sensitivity and specificity in the biochemical group (Figure 3). The sum of sensitivity and specificity for the GAG studies are just statistically significant with 95 percent confidence limits of 1.06 to 1.52. Table 3 gives the frequency distribution of the results of the GAG test for employees with normal and abnormal biochemical results. The distributions differ in their spread (variance) and not in their location (means). Finally, the correlation matrix for the four tests are given in Table 4. |t There is no significant correlation, as measured by between any pair of the screening tests. This is also true when they are sorted by biopsy status. After reviewing the results, a reclassification of the 51 employees who had biopsies was assessed in regard to whether there was chemically induced liver damage. Ultrasonographic evaluation was reclassified by Or. Taylor and the liver biopsies by Drs. Tamburro and Popper. Table 5 gives the results of this additional analysis which demonstrates that there was no agreement that could not be explained by chance (P = 0.60). CMA 003700 6 Discussion The increasing industrialization in highly developed Western countries, such as the United States, continues to provide concern, not only for the health and safety of the industrial workers, but also for the surrounding conmunities in the areas of these industries. It is highly desirable to identify and validate the reliability of screening and diagnostic techniques which will identify the early development of injury due to the exposure of a variety of chemicals such as vinyl chloride. The assessment of newly developing techniques on a high-risk, exposed worker population, who have been carefully screened and prospectively followed, provide the most reliable method for determing both the sensitivity and specificity of these technical procedures in the asymptomatic subclir.ical high- risk exposed coimunity population. The failure of such techniques to provide sufficient sensitivity in the presence of a required specificity is of critical clinical importance. This is especially so where the incidence of disease is relatively low and the population exposed large. Tests which provide a high sensitivity but of low specificity can and do medically stigmatize the population under surveillance leading to unnecessary anxiety and socioeconomic disturbances which can far outweigh the benefit of early detection of even serious disease in a smaller population. Far too often screening techniques which have been developed in a highly diseased, clinically overt, hospitalized population are applied to an asymptomatic, clinically well-working populations without adequate determination of the sensi tivity and specificity at this earlier stage of disease development. In this study, all four techniques had, in the highly diseased hospitalized population, demonstrated either a sensitivity or specificity suggestive for the identification of underlying chemically related liver disease. Some techniques (Maricq and Kupchella) appeared ideal for community studies since they were non- invasive, relatively inexpensive, and provided a means of screening which would be highly accepted by a conmunity. CMA 003701 7 This prospectively designed study has allowed us to estimate the ability of ultrasonography, nailbed capillary assessments, radioisotopic scanning, and glycosaminoglycan excretions to correctly predict the presence and absence of hepatic disease as documented by an exposed population. These studies clearly show that none of the four screening techniques sufficiently agree with either the biopsy, the biochemical results, or each other in a well defined population; they do not provide sufficient sensitivity or specificity to be useful as early indicators of chemical exposure injury. The inclusion of nine individuals with intermediate biochemical results, who were originally misclassified as abnormal, would decrease the sum of sensi tivity and specificity in all three screening tests. With their exclusion only the GAG tests provided results better than might be expected by chance (P< 0.05). Even the GAGs, from a pragmatic point of view, provide too high a false positive rate to be useful in its present stage. Possibly, with increased refinement and further study, this might provide a simple non-invasive technique for the identification of increased collagen changes related to chemical injury. More immediately, it should be duplicated to rule out chance. Had we eliminated the four employees with an abnormally low biochemical test values, the sum of sensitivity and specificity for the GAGs and nailbed skin capillary screening tests would have been slightly reduced and that for the scan slightly increased. The GAG would still be of bordering significant (X^ = 3.73), and the scan not significant (x| = 1.46). Finally, the tests not only disagree with each other, but within the biopsy group there was no agreement between the biopsies and biochemical results. It should be noted, however, that the biochemical studies used in analysis were those chronologically closest to September, 1977, and not to the date of biopsy. CMA 003702 / s i: ;::ively c = 3'v,=d study has allowed ,j ;c esv:"3;a c.-.= t" / .-a: :-ograpny , nail bed catiilary assessments, raaia sc-tcpic scanning, and g'ycosaminoglycan excre';nns to correctly predict the presence and absence of hepatic disease as documented by an exposed population. These studies clearly snow that none of the four screening techniques sufficiently agree with either the biopsy, the biochemical results, or each other in a well defined population; they do not provide sufficient sensitivity or specificity to be useful as early indicators of chemical exposure injury. The inclusion of nine individuals with intermediate biochemical results, who were originally misclassified as abnormal, would decrease the sum of sensi tivity and specificity in all three screening tests. With their exclusion only the GAG tests provided results better than might be expected by chance (P< 0.05). Even the GAGs, from a pragmatic point of view, provide too high a false positive rate to be useful in its present stage. Possibly, with increased refinement and further study, this might provide a simple non-invasive technique for the identification of increased collagen changes related to chemical injury. More iimediately, it should be duplicated to rule out chance. Had we eliminated the four employees with an abnormally low biochemical test values, the sum of sensitivity and specificity for the GAGs and nailbed skin capillary screening tests would have been slightly reduced and that for the scan slightly increased. The GAG would still be of bordering significant (X^ = 3.73), and the scan not significant (x| = 1.46). Finally, the tests not only disagree with each other, but within the biopsy group there was no agreement between the biopsies and biochemical results. It should be noted, however, that the biochemical studies used in analysis were those chronologically closest to September, 1977, and not to the date of biopsy. CMA 003703 8 The test determinations and the biopsies may have been as long as three years apart. An analysis of the biochemical tests value done at the time of the biopsy would have more accurrately reflected the liver status, as shown by histology (15). It has been shown, in previously published studies, that biochemical and histological findings each correlate with chemical injury and chemical exposure (16, 17). CMA 003704 9 References 1. Maltoni, C. and Lefemine, G. Carcinogenicity to bioassays of vinyl chloride I. Research plan and early results. Environmental Perpectives, 7:387-405, 1974. 2. Creech, J. L. and Johnson, M. N, Angiosarcoma of the liver in the manufac ture of polyvinyl chloride. Journal of Occupational Medicine, 16:150-151, 1974. " 3. Taylor, K. J. W., Williams, D. M. J., Smith, P. M. and Dach, B. W. Grey scale ultrasonography for monitoring industrial exposure to hepatotoxic agents. Lancet, 1:1222-1224, 1975. 4. Whelan, J. G. Jr., Creech, J. L. and Tamburro, C. H. Angiographic and iso topic characteristics of hepatic angiosarcoma found in vinyl chloride workers. Radiology, 118:549-557, March 1976. 5. Maricq, H. R., Johnson, M. N., Whitstone, C. L. and LeRoy, E. C. Capillary abnormalities in polyvinyl chloride production workers. JAMA, 236, 1368- 1371, 1976. ----- 6. Kupchella, C. E. and Tamburro, C. H. Urinary and tissue glycosaminog!yean patterns in angiosarcoma and other vinyl chloride exposure - associated liver injury. In Prevention and Detection of Cancer, Part I, 1:915-926, ed. Niebergs, H., Marcel Dekker, Inc., 1977. 7. Taylor, K. J. W., Carpenter, D. A., Hill, C. R. and McCready, V. R. Grey scale ultrasound imaging the anatomy and pathology of the liver. Radiology, 119: 415-423, 1976. 8. Maricq, H. R. and LeRoy, E. C. Patterns of finger capillary abnormalities in connective tissue disease by wide field microscopy arthritis pheum, 16: 619-629, 1973. ~ 9. Curran, K. L., Kupchella, C. E. and Tanburro, C. H. Urinary glycosaminoglycan patterns in angiosarcoma of the liver. Cancer, 40:3050-3053, 1977. 10. Whelan, J. G. Jr., Greenberg, R. and Tamburro, C. H. The effectiveness of radioisotopic scans and grey scale ultrasonography in. the detection of liver damage. Gastroenterology, 79:1129, 1980. 11. Rosch, J., Anconovic, R. and Dotter, C.T. Transjugular approach to the liver, biliary system and portal circulation. American Journal of Roentgenology, 125:602-608, 1975. 12. Whelan, J. G. Jr., Creech, J. L. and Tanburro, C. H. Primary liver cancer detection in vinyl chloride workers by radioisotopic scanning (In Preparation) 13. Taylor, K. J. W,, Glees,J. P., Smith, T. A. and Carpenter, D. A. Ultrasonic examination of the liver. In Ultrasound in Medicine, Vol. .2, pp. 173-174 (Eds.) White, D. N. and Barnes, R., Plenum Press, New York, 1976. CMA 003705 1. Maltoni, C. and Lefemine, G. Carcinogenicity to bioassays of vinyl chloride I. Research plan and early results. Environmental Perpectives, 7:387-405, 1974. 2. Creech, J. L. and Johnson, M. N. Angiosarcoma of the liver in the manufac ture of polyvinyl chloride. Journal of Occupational Medicine, 16:150-151, 1974. 3. Taylor, K. J. W., Williams, 0. M. J., Smith, P. M. and Dach, B. W. Grey scale ultrasonography for monitoring industrial exposure to hepatotoxic agents. Lancet, i_:1222-12?4, 1975. 4. Whelan, J. G. Jr., Creech, J. L. and Tamburro, C. H. Angiographic and iso topic characteristics of hepatic angiosarcoma found in vinyl chloride workers. Radiology, 118:549-557, March 1976. 5. Maricq, H. R., Johnson, M. Whits tone, C. L. and LeRoy, E. C. Capillary abnormalities in polyvinyl chloride production workers. JAMA, 236, 1 368- 1371, 1976. ---- 6. Kupchella, C. E. and Tamburro, C. H. Urinary and tissue glycosaminog!yean patterns in angiosarcoma and other vinyl chloride exposure - associated liver injury. In Prevention and Detection of Cancer, Part I, 1:915-926, ed. Niebergs, H., Marcel Dekker, Inc., 1977. 7. Taylor, K. J. W., Carpenter, D. A., Hill, C. R. and McCready, V. R. Grey scale ultrasound imaging the anatomy and pathology of the liver. Radiology, 119: 415-423, 1976. 8. Maricq, H. R. and LeRoy, E. C. Patterns of finger capillary abnormalities in connective tissue disease by wide field microscopy arthritis pheum, 16: 619-629, 1973. -- 9. Curran, K. L., Kupchella, C. E. and Tanburro, C. H. Urinary glycosaminoglycan patterns in angiosarcoma of the liver. Cancer, 40:3050-3053, 1977. 10. Whelan, J. G. Jr., Greenberg, R. and Tamburro, C. H. The effectiveness of radioisotopic scans and grey scale ultrasonography in. the detection of liver damage. Gastroenterology, 79:1129, 1980. 11. Rosch, J., Anzonovic, R. and Ootter, C.T. Transjugular approach to the liver, biliary system and portal circulation. American Journal of Roentgenology, 125:602-608, 1975. 12. Whelan, J, G. Jr., Creech, J. L. and Tanburro, C. H. Primary liver cancer detection in vinyl chloride workers by radioisotopic scanning (In Preparation). 13. Taylor, K. J. W,, Glees,J. P., Smith, T. A. and Carpenter, 0. A. Ultrasonic examination of the liver. In Ultrasound in Medicine. Vol. Z_, pp. 173-174 (Eds.) White, D. N. and Barnes, R., Plenum Press, New York, 1976. CMA 003706 10 14. Taylor, K. J. W. and Carpenter, D. A. Comparison of radioisotopic and ultra sound examination in the investigation of hepatobiliary disease. In Ultrasound in Medicine, Vol. 1, pp. 159-167 (Ed.) White, D. N., Plenum Press, New York", 1976. 15. Clarmont, R.J. and Chalmers, T.C. The transaitiinase tests in liver disease. Medicine, 46:197-207, 1967. 16. Tamburro, C.H. and Greenberg, R. Effectiveness of federally-required medical laboratory screening in the detection of chemical liver injury. Environmental Perspectives, 1980. In Press. 17. Tamburro, C.H. and Greenberg, R.A. Identification of human toxicity and carcinogenicity by ethylene derivatives in mechanisms of toxicology and hazard evaluation (eds. Holmstedt, B., et al) Elsevier/North-Holland Biomedical Press, New York, New York, pp. 319-334, 1980. 18. Tamburro, C.H., Makk, L. and Popper, H. Early hepatic histological alterations among chemical (vinyl monomer) workers. Gastroenterology, 77:A43, 1979. CMA 003707 Table i CLASSIFICATION Biopsy Biochemical Ultrasound Microvascular DETERMINED BY CRITERIA 1) Dr. Popper, Pathologist a) Positive by consensus agreement if medically significant pathology 2) Dr. Makk, Pathologist is present. Negative otherwise. 3) Dr. Tamburro, Hepatologist b) Pathology is of chemical or non chemical origin. (18) Liver "Function" Tests Group 1 Group 2 SGPT GGTP ICG BILIRUBIN SGOT ALK. PHOSPHATASE Positive if two or more Group tests were abnormal or if one Group 1 and both Group 2 tests were abnormal. Negative if all six tests were normal. Intermediate otherwise. Dr. Taylor Defined as negative if the over all impression was normal. Posi tive otherwise. Dr. Maricq Defined as negative if there were no microvascular abnormalities. Positive otherwise. Glycosaminoglycans Ur_o__m c _ UG Uronic Acid acid MG C_ rea,t.ini.ne Defined as positive for values less than 2.0 or greater than 4.8. Negative otherwise. Liver Scan Dr. Whelan Defined as positive if any patho logical defect was detected. Negative Otherwise. 003708 u L cUJJLe i CLASSIFICATION Biopsy Biochemical Ultrasound Microvaucular DETERMINED BY CRITERIA 1) 2} 3) Dr. Popper, Pathologist Dr. Makk, Pathologist Dr. Tamburro, Hepatologist a) Positive by consensus agreement if medically significant pathology is present. Negative otherwise. b) Pathology is of chemical or non chemical origin. (18) Liver"Function" Tests Group 1 Group 2 SGPT GGTP TCG BILIRUBIN SCOT ALK. PHOSPHATASE Positive if two or more Group tests were abnormal or if one Group 1 and both Group 2 tests were abnormal. Negative if all six tests were normal. Intermediate otherwise. Dr. Taylor Defined as negative if the over all impression was normal. Posi tive otherwise. Dr. Maricq Defined as negative if there were no microvascular abnormalities. Positive otherwise. 1ycosaminoglycans Uronic _ UG Uronic Acid ncid MG C,, rea. ti,ni,ne Defined as positive for values less than 2.0 or greater than 4.8. Negative otherwise. Liver Scan Dr. Whelan Defined as positive if any patho logical defect was detected. Negative Otherwise. TABLE 2 COMPARISON OF BIOPSY AND BIOCHEMICAL DETERMINATIONS OF THE PRESENCE OF LIVER DISEASE 12 BIOPSY BIOCHEMICAL POSITIVE 'NEGATIVE SUM BIOCHEMICAItY TOTAL INDETERMINATE POSITIVE NEGATIVE SUM 3 0 3 18 21 88 26 29 r - 0.20966 X^ = 0.200 N.S. Matched 1 = (l8 " 2 /l8 =16.1 P < 0.001 15 7 22 36 15 51 CMA 003710 TABLE 3 FREQUENCY (F) AND RELATIVE FREQUENCY (R.F.) OF GAGS FOR NORMAL AND ABNQRflAL EIOCHEMICAL RESULTS GAG <2 2<3 3<4 4<5 5+ Sum Mean Variance Normal f R.F. 4 0.111 16 0.444 13 0,361 1 0.028 2 0.056 36 1.000 2.886 0.746 Abnormal f R.F. 6 0.250 7 0.292 6 0.250 1 0.042 4 0.167 24 1.000 3.160 1.776 t = 0.968 N.S. 5a F = 2.381 P < 23, 35 T Test Independent Means 0.05 F Test Independent Variances ( Two Taile' CMA 003711 FRZOUINCY (F) AND RELATIVE F?.ZCDZ'TCY (R.F CF GAGS FOR NORMAL AND A3N0R-'LAL GAG <2 2<3 3<4 4<5 5+ Sum Mean Variance Normal * R.F. 4 0.111 16 0.444 13 0.361 1 0.028 2 0.056 36 1.000 2.886 0.746 Aic, bnormaal . r, , 6 0.250 7 0.292 6 0.250 1 0.042 4 0.167 24 1.000 3.160 1.776 t = 0.968 N.S. T Test Independent Means si F = 2.381 P < 0.05 F Test Independent 2 3/35 Variances ( Two Taile CMA 003712 14 TABLE 4 CORRELATION MATRIX (Ro) BETWEEN FOUR SCREENING TESTS USED TO PREDICT THE PRESENCE OR ABSENCE OF LIVER DISEASE GAG CAPILLARY ANALYSIS ASSESSMENT GAG ANALYSIS 0.08 CAPILLARY ASSESSMENT ULTRASOUND STUDY (113) (87) (84) RADIOISOTOPIC SCAN (120) (114) ULTRASOUND STUDY -0.03 0.03 (88) RADIOISOTOPIC SCAN 0.005 0.07 0.06 * None of the correlations are statistically significant (a = 0.05). The correlations are given above the diagonal. The sample size is given in parenthesis below the diagonal. CMA 003713 TABLE 5 RECLASSIFICATION OF BICPSIED EMPLOYEES FOR CHEMICALLY INDUCED ABNORMALITIES Biopsy Ultra Sound Chemical Abnormality Other Chemical Abnormality Other 7 26 5 13 SUM 33 18 SUM 12 39 51 Specificity =* 13/39 = 0.333 Sensitivity = 7/12 = 0.583 Sum = 0.916 CMA 003714 >0 TABLE 5 KECLASStFICATTCW CF BICPSIED E-'PLOYEES FOR CHEMICALLY INDUCED ABNOIMALITIES Biopsy Ultra Sound Chemical Abnormality Other Chemical Abnormality Other 7 26 5 13 SUM 33 18 SUM 12 39 51 Specificity = 13/39 = 0.333 Sensitivity = 7/12 = 0.583 Sum " 0.916 CMA 003715 GROUP A FIGURE 1 GKCUP B *Not seen by Doctor Taylor CMA 003716 sensitivity and specificity for four screening TESTS FOR THE PREDICTION OF LIVER ABNORMALITIES (AS DETERMINED BY BIOPSY) o H o0) ft CO T) Crt S i>i +-> CO +J *H CCQ 0) CO \ 1 Nunrber Employees Positive Biopsies Negative Biopsies 15 15 15 15 CMA 0 0 3 7 1 7 >* 4J rf u H fl-f r-t Voo* ln c; CTj L;.)) ] r4 WK <\) lO 0 Niinhr*r Hrployees I>v;i 1 ho Biopsies Biopsies SENSITIVITY AMD SPECTFICITY FOR FOUR SCREENING TESTS FOR THE PREDICTION OF LIVER ABNORMALITIES (AS DETERMINED BY BIOPSY) (51) 36 13 (^9) 34 15 (51) 36 15 (51) 36 15 nr- g o u CMA 0 0 3 7 1 9 Figure 3 SENSITIVITY AND SPECIFICITY FOR FOUR SCREENING 05 TESTS FOR THE PREDICTION OF LIVER ABNORMALITIES (AS DETERMINED BY REVIEWED BIOCHEMICAL TESTS) 1.29 Sum: Specificity plus sensitivity 1.16 95% Limits 1.15 Sensitivity 0 Nvirber Employees Positive Biochemical Negative Biochemical Specificity GAG Microvascular Ultrasound Scan (60) 24 36 (57) 22 35 (37) (61) 24 37 10 STUDIES ON THE MUTAGENICITY OF VINYL CHLORIDE METABOLITES AND RELATED CHEMICALS A. D. Laumbach, S. Lee, J. Wong, and U. N. Streips Department of Microbiology and Immunology University of Louisville School of Medicine Department of Chemistry Louisville, Kentucky 40201 I. INTRODUCTION The studies by Viola et al (26) and Maltoni et al (15) established the carcinogenic potential of vinyl chloride monomer. The detection of angiosarcoma in industrial workers exposed to polyvinyl chloride suggested a causal relationship between this chemical and the development of hepatic abnormalities (4,12). Hefner et al (7) have delineated the metabolic fate of inhaled vinyl chloride in rats and proposed that the epoxide, chlorooxirane, and chloroacetaldehyde were the carcinogenic intermediates. Their hypothesis has been supported by the work of several laboratories (3,14,16,19, Elmore, Wong, Laumbach and Streips, submitted for publication) using bacterial strains as mutagenic indicators. In this communication v.re present additional data concerning the mutagenicity and the potential mechanisms of action of several vinyl chloride metabolites, including the previously unreported chloroacetadehyde monomer hydrate, chloroacetaldehyde dimer hydrate, and chloroacetaldehyde trimer. Epichlorohydrin, a mutagenic/carcinogenic (21,25) methylene homolog of chlorooxirane was also examined.II. II. PROCEDURES AND MATERIALS USED A. Bacterial Strains The bacterial strains utilized in these studies are presented in Table I. The Bacillus subtilis strains were all maintained on AK agar (BBL). Salmonella typhimurium cultures were obtained from B. N. Ames (l) and were stored on Nutrient Agar (Difco) plus 5g NaCl per liter. CMA 003720 Laumbach, A.D. , Lee, S. , Wong, J. , and Streips, U.N. B. Mutagenicity Assays The indirect assay utilized repair deficient strains of B. subtilis. The procedure was a modification of the "rec-assay" described by hada et al (9). Cells were grown overnight in Nutrient Broth (Difco) at J7C in a rotary incubator shaker, then diluted tenfold in phosphate buffer (pH 7.0). The suspended cultures were streaked onto Nutrient Agar plates (Difco). Filter paper discs (6 mm) were saturated with the chemical solutions to be examined, then were placed onto the agar plates next to the streaked bacterial cultures. Following incubation at 37C overnight the plates were examined and the lethality and mutagenic potential of the test chemicals were assessed by comparing inhibition zones between the B. subtilis 168 wild type, a repair-capable strain and the various DNA repair-deficient strains. In all these studies 4-nitroquinoline-l-oxide (4NQ0) was used as the positive mutagenic control. Direct mutagenicity assays utilized the S. typhimurium tester strains described by Ames (l). The chemicals were examined by the methods of McCann et al (16). The cultures were grown in Nutrient Broth plus 0.5# NaCl overnight in a rotary incubator shaker at 37C. A mixture of the test chemical (0.1 ail) in dimethyl sulfoxide (DM50) and 2 ml of soft agar (0.6# agar, 0.6# NaCl, 0.5 mM biotin, and 0.5 mM histidine) was added to 0.1 ml of the bacterial culture. The solutions were mixed thoroughly and overlaid onto minimal plates [Vogel-Bonner E medium (27), 1.5# agar, and 2# glucose]. Control samples were prepared by omitting the test chemicals. For the positive mutagenesis control, 4NQ0 was added to the mixtures in place of the test chemicals. All plates were incubated for 48 hr at 37C prior to the enumeration of revertant colonies. C. Chemical Compounds The chemical compounds utilized in these studies were prepared, purified, and analyzed by previously reported techniques (Elmore, Wong, Laumbach, and Streips, submitted for publication). D. Preparation of DNA Transforming DNA was isolated from B. subtilis cultures by the method described by Young and Wilson (29T- In some of the experiments the cultures were pretreated for 15 min either with chloroacetaldehyde (16 mM) or epichlorohydrin (16 mM) prior to the extraction procedure. In alternate experiments S-9 liver homogenate mix was added to the compounds prior to addition to bacteria. The S-9 liver homogenate contains per ml, 0.3 ml of the S-9 fraction, 8 mM MgCl2 33 mM KC1, 5 mM glucose-6-P, 4 mM NADP, and 100 mM sodium phosphate (pH 7.4). The DNA concentration in all lysates were assayed by the method of Richards (20). 003721 E. Treatment of DNA In vitro with Chemicals A sample of B. subtilis transforming DNA (0.9 ml) in standard saline citrate (SSC) (0.15 M MaCl-0.015 M trisodium citrate, pH 7.0) was combined with 0.1 ml chloroacetaldehyde (1.0 M in DMS0) or 0.1 ml u STUDIES ON THE MUTAGENICITY OF VINYL CHLORIDE METABOLITES epichlorohydrin (1.0 M in DMSQ). The mixture was allowed to react for 1 hr v. Lth occasional shaking. Following this treatment the treated DMA was dialyzed at OC against three 500 ml changes of SSC for 24 hrs. In alternate experiments the DNA-chemical mixtures were placed in a dialysis bag and immersed in the S-9 liver homogenate mix. These samples were dialyzed in SSC as above. F. Competent Cultures for Transformation Assays The procedures for the development of competence v/ere similar to those described (23). B_. subtilis cells were grown in a modified Spizizen's minimal medium (GMIJ (29) for 90 min at 37C after cessation of logarithmic growth in a rotary incubator shaker. The cells were then diluted tenfold into GMII medium (29) and incubated for an additional 60 min at 37C in the shaker. At this time the culture has attained maximum competence. G. Transformation Procedures A sample (0.1 ml) of extracted, treated or untreated DNA was added to 0.8 ml of the competent cultures and incubated at J7G for 30 min in the shaker. The reaction was terminated by the addition of 0.1 ml of deoxyribonuclease (500 yg/ml, Worthington Biochem. Corp.) for 15 min at 37C. The cells were plated on appropriate selective minimal media and incubated at yiC for 48 hrs. III. RESULTS A summary of preliminary mutagenesis screening experiments with potential vinyl chloride monomer metabolites and related compounds is presented in Table II. It is evident that chlorooxirane and chloroacetaldehyde are the ultimate mutagens in this system. These results agree with the published data (3,16). In addition, this table describes the mutagenicity of the other chemical forms of chloroacetaldehyde, not ably a monomer hydrate, a dimer hydrate, and a trimer. The hydrate and dimer hydrate forms have been shown to form an equilibrium mixture by the spontaneous rearrangement of chloroacetaldehyde under physiological conditions (Elmore, Wong, Laumbach, and Streips, submitted for publication),and these hydrate forms must be regarded as potential metabolites of consequence. Purified dimer hydrate and trimer were synthesized under laboratory conditions. Neither acetaldehyde, chloroacetic acid, nor chloroethanol showed a significant level of mutagenicity in these assays. Other investigators have reported the mutagenicity of chloroethanol, however, either high concentrations or activation with microsomal enzymes was required for activity (3,16). Our results agree with those of McCann et al (16). These experiments suggest a molecular relationship involving the proximity of the chloride group to the aldehyde moiety for mutagenic activity. In this regard we are currently examining structurally analogous ketones, substituted with various halogens. Epichlorohydrin (l-chloro-2,3 epoxypropane) was also mutagenic in screens using the Salmonella tester strain TA100. r'V.'.-taCi.C'L o_1 - i 3.16 air.st thraa ^CC .71 ol -- .er.t s the DNA-chemicai mixtures v;ere placed in a dialysis bag ar.d i: zzersed in the 5-9 liver hoc.oger.ate mix. These samples were disJLyzsd .r. 55C as above. F. Competent Cultures for Transformation Assays The procedures for the development of competence were similar to those described (23). B. subtilis cells were grown in a modified Spioizen's minimal medium (GI.il) (29) for 90 min at 370 after cessation of logarithmic growth in a rotary incubator shaker. The cells were then ill_:ei oenfold into 2.Z.Z medium (29) and incubated for an additional 60 min at 370 in the shaker. At this time the culture has attained maximum ccmtezenoe. G. Transformation Procedures A sample (0.1 ml) of extracted, treated or untreated DNA -was added to O.S ml of the competent cultures and incubated at 37C for 30 min in the shaker. The reaction was terminated by the addition of 0.1 ml of deoxyribonuclease (500 yg/ml, Worthington Biochem. Corp. ) for 15 min at 37C. The cells were plated on appropriate selective minimal media and incubated at 370 for 18 hrs. Ill. RESULTS A summary of preliminary mutagenesis screening experiments with potential vinyl chloride monomer metabolites and related compounds is presented in Table II. It is evident that chlorooxirane and chloroacetaldehyde are the ultimate mutagens in this system. These results agree with the published data (3>16). In addition, this table describes the mutagenicity of the other chemical forms of chloroacetaldehyde, not ably a monomer hydrate, a dimer hydrate, and a trimer. The hydrate and dimer hydrate forms have been shown to form an equilibrium mixture by the spontaneous rearrangement of chloroacetaldehyde under physiological conditions (Elmore, Wong, Laumbach, and Streips, submitted for publication),and these hydrate forms must be regarded as potential metabolites of consequence. Purified dimer hydrate and trimer were synthesized under laboratory conditions. Neither acetaldehyde, chloroacetic acid, nor chloroethanol showed a significant level of mutagenicity in these assays. Other investigators have reported the mutagenicity of chloroethanol, however, either high concentrations or activation with microsomal enzymes was required for activity (3*16). Our results agree with those of McCann et al (16). These experiments suggest a molecular relationship involving the proximity of the chloride group to the aldehyde moiety for mutagenic activity. In this regard we are currently examining structurally analogous ketones, substituted with various halogens. Epichlorohydrin (l-chloro-2,3 epoxypropane) was also mutagenic in screens using the Salmonella tester strain TA1QQ. CMA 003723 Laurcbach, A. D.,Lee, S., Wong, J., and Streips, U. N. Further experiments examined the effect of proposed metabolites on several different DNA repair deficient strains of B_. subtilis. Chlorooxirane and the different forms of chloroacetaldehyde were all found to specifically inhibit the growth of strain MC-1, which lacks recombination repair (17) (Table III). Epichlorohydrin was capable of moderate reactivity only in the presence of the S-9 fraction. Quantitative mutagenesis assays with Salmonella strain TA1Q0, an indicator for base-pair substitution mutations, revealed that chloro acetaldehyde monomer had the highest mutagenic capacity of all the reactive metabolites (Table IV). The monomer-dimer hydrates, dimer hydrate, and trimer show progressively decreasing mutagenic efficiency as evidenced by the higher chemical concentration required for eliciting maximum reversion. All forms of chloroacetaldehyde were very toxic, thus the mutagenic response of each compound was limited to a narrow range of concentrations. However, epichlorohydrin, a weak mutagen by comparison, has a broad mutagenic spectrum and a corresponding low toxicity. Since the mutagenic activity of the compounds constituted strong evidence that DNA was a primary target of attack, we examined the inter action of chloroacetaldehyde and epichlorohydrin with transforming DNA. It is known that the biological activity of transforming DNA can be altered by exposure to physical and chemical agents (8,22). Previous studies have shown that chloroacetaldehyde can bind to DNA in vitro (11). Accordingly, transforming DNA isolated from 13. subtilis 168WT was treated with either chloroacetaldehyde or epichlorohydrin as described in Materials and Methods. The treated DNA was examined in transformation assays utilizing several different auxotrophic strains of B. subtilis as the recipients. Data presented in Table V reveals that in vitro treatment of DNA with either compound has little or no apparent effect on the biological activity of this DNA in transformation. Since both chloroacetaldehyde and epichlorohydrin demonstrated mutagenic activity in the Salmonella TA100 strain, we examined the effect of these two compounds on B. subtilis DNA in vivo. Transforming DNA was isolated from B. subtilis following a 15 min exposure to the mutagenic chemicals. The DNA concentration was calculated from these samples, and levels equivalent to those used in the in vitro assays were added to competent cultures. The results of these transformation assays are shown in Table VI. Two major effects are evident with chloroacetaldehyde in vivo treated DNA. First, there was a major depression of the biological activity in the transforming DNA. Secondly, the depression showed genetic marker specificity. Moreover, the DNA segments containing genetic markers which have previously been shown to be associated to macromolecular structures such as the cell membrane (6,24,28) or the cell wall (Streips, Doyle, Sueoka, Brown, and Fan, submitted for publication) were selectively protected from attack by chloroacetaldehyde and epichlorohydrin. The activity of epichlorohydrin was less in these experiments, however, the patterns of specific marker inactivation are quite similar. The addition of the S-9 mix to the chemicals prior to addition to the cells, did not cause significant alteration in trans formation efficiency (results not shown). In some samples there was an effect on the transforming DNA by DMSO, therefore all transformation values were corrected to account for this parameter. CMA 003724 STUDIES ON THE MUTAGENICITY OF VINYL CHLORIDE METABOLITES IV. DISCUSSION The major findings reported in this manuscript can be summarized: 1) We have confirmed the mutagenicity of chloroacetaldehyde and chlorooxirane, and extended it to include the additional potential metabolites, chloroacetaldehyde monomer hydrate, dimer hydrate and trimer, as well as the previously unreported chlorooxirane homolog, epichlorohydrin. 2) We have shown that recombination repair appears to be the mechanism for the correction of vinyl chloride metabolite elicited damage. 3) Chloro acetaldehyde causes a decrease in the biological activity of transforming DNA only if the cells are treated with the mutagen prior to the extraction of the DNA. In vitro studies showed no effect. 4) Epichlorohydrin apparently differs markedly from the vinyl chloride metabolites in mutagenic activity. To understand the mutagenic potential of an environmental carcinogen, such as vinyl chloride and related chemicals,it is necessary to determine both its metabolic fate and probable mechanism of action for alteration of cellular processes. This report, as well as others, (3,14,16) has identified the potential active metabolites in vinyl chloride monomer mediated carcinogenesis. Furthermore, on the basis of a series of studies in microbial systems, we can postulate probable mechanisms of action of the vinyl chloride monomer metabolites and related chemicals. Under standing of these mechanisms is necessary for the development of possible blocking agents to the carcinogenic activity. Recombination repair appears to be induced to correct DNA lesions caused by vinyl chloride monomer metabolites and epichlorohydrin. Salmonella strain TA100 which lacks excision repair (uvr"), yet retains the capacity for recombination repair is capable of recovery and can express mutation following exposure. Furthermore, experiments with several repair-deficient B. subtilis mutants demonstrate that only the recombination repair mutant is specifically sensitive to the active metabolites, whereas the excision repair mutants and the wild type strain are relatively unaffected. The nature of the lesions may specifically evoke the recombination repair mechanism (10), or, alternatively, the chemical reactivity of the metabolites may directly suppress other repair. It is known that recombination repair is inducible, while other types of repair are mostly constitutive (5). Since chloroacetaldehyde has been shown to specifically interact with proteins containing -SH groups (J. Hoffman, personal communication), it is possible that the chemical could inactivate the constitutive repair enzymes leaving the repair to an inducible system. The requirement for recombination repair of damage induced by these chemicals suggests the potential route of mutagenesis in bacteria. Recombination repair has been shown to be error prone (16). In this sense it resembles postreplication repair in mammalian cells (13). Thus, we can postulate that the analogous error prone repair pathway, postreplication repair, may function in mammalian cells in response to vinyl chloride metabolite elicited damage. A relationship between postreplication repair caused errors and somatic mutation and carcinogenesis has been suggested in patients with the skin disease, xeroderma pig mentosum (13)* The rajcr :ir.iir.es r^rrr'.ei ir. *.hir .varus criy. car. be summarised: 1) V.'e have car.: irmad are- mutagenicity ef ehlcroacetaldehyde and ehlereoxirur.e, ar.d extended it dr. irtluis the additional potential metabolites, chleroaceealdehyde ncr.or.er hydrate, diner hydrate and triner, as 7.-211 as have shot/n that recombination repair appears to be the mechanism for the correction of vinyl chloride metabolite elicited damage. 3) Chlcroacetaldehyde canoes a decrease ir. the biological activity of transforming DMA only if the cells are treated with the mutagen prior to the extraction In vitro studies showed no effect. 4) Epichlcrohydrin lie r.etasoiites umerstars su vinyl c: "t ^ * * * :ter.tial of an environmental carcinogen, chemicals,it is necessary to determine both its metabolic fate and probable mechanism of action for alteration of cellular prccesses. This report, as well as others, (3,14,16) has identified the potential active metabolites in vinyl chloride monomer mediated carcinogenesis. Furthermore, on the basis of a series cf studies in microbial systems, we can postulate probable mechanisms of action of the vinyl chloride monomer metabolites and related chemicals. Under standing of these mechanisms is necessary for the development of possible blocking agents to the carcinogenic activity. Recombination repair appears to be induced to correct DM lesions caused by vinyl chloride monomer metabolites and epichlorohydrin. Salmonella strain TA1Q0 which lacks excision repair (uvr"), yet retains the capacity for recombination repair is capable of recovery and can express mutation following exposure. Furthermore, experiments with several repair-deficient 3. subtilis mutants demonstrate that only the recombination repair mutant is specifically sensitive to the active metabolites, whereas the excision repair mutants and the wild type strain axe relatively unaffected. The nature of the lesions may specifically evoke the recombination repair mechanism (10), or, alternatively, the chemical reactivity of the metabolites may directly suppress other repair. It is known that recombination repair is inducible, while other types of repair are mostly constitutive (5). Since chloroacetaldehyde has been shown to specifically interact with proteins containing -SH groups (J. Hoffman, personal communication), it is possible that the chemical could inactivate the constitutive repair enzymes leaving the repair to an inducible system. The requirement for recombination repair of damage induced by these chemicals suggests the potential route of mutagenesis in bacteria. Recombination repair has been shown to be error prone (16). In this sense it resembles postreplication repair in mammalian cells (13)- Thus, we can postulate that the analogous error prone repair pathway, post replication repair, may function in mammalian cells in response to vinyl chloride metabolite elicited damage. A relationship between post replication repair caused errors and somatic mutation and carcinogenesis has been suggested in patients with the skin disease, xeroderma pig mentosum (13). CMA 003726 Laumbach, A. D., Lee, S., Wong, J., and Streips, U. N. The increased inhibitory activity of the chloroacetaldehyde diir.er and trimer forms for the other repair-deficient B_. subtilis strains (Table III) may have been nonspecific killing of the cells, since all the strains other than MC-1 showed identical levels of inhibition. The necessity for metabolic activation of epichlorohydrin could reflect either a lack of permeability of the nonactivated compound or the requirement of a metabolite of this compound as the true mutagenic species. Neither chloroacetaldehyde nor epichlorohydrin seemed to affect transforming DNA in vitro (Table V). Although several investigators have reported that CAA specifically modifies bases and causes mismatched base pairs (2,11), this reaction in vitro does not seem to affect the biological activity of the DNA. In contrast, DNA which was isolated from cells treated with either chloroacetaldehyde or epichlorohydrin (in vivo, Table VI) was severely affected. The overall biological activity of the transforming DNA is depressed, and it appears that the regions of the genome which are not protected by either the cell membrane or cell wall are most susceptible to attack and inactivation. It has also been postulated both in Escherichia coli and ]3. subtilis that the replication origin, terminus, and replication fork are all outer surface bound (18,24). Thus, these would be protected regions from chloroacetaldehyde attack and the nonreplicating DNA in the cytoplasm would be most susceptible. In this connection, recent experiments in our laboratory (Laumbach, Lee, Wong, and Streips, manuscript in preparation) have shown that chloroacetaldehyde causes enhanced mutation levels in cultures with nonreplicating genomes. This may imply that chloroacetaldehyde could be active in mammalian cells during growth stages where little DNA synthesis occurs. The mode of action of epichlorohydrin, a known carcinogen (25), differs from that of the vinyl chloride monomer metabolites. Although epichlorohydrin causes similar base substitution mutations in Salmonella tester strain TA 100, it is a comparatively weaker alkylating agent based on quantitative assay. Epichlorohydrin also exhibits a lower toxicity level than vinyl chloride monomer metabolites, thus epichloro hydrin can demonstrate mutagenic activity through a wider range of concentrations. In addition, our laboratory has preliminary evidence that epichlorohydrin produces higher levels of mutation in Salmonella cultures which are actively replicating DNA than in cultures which have been arrested in DNA replication (Laumbach, Lee, Wong, and Streips, manuscript in preparation). The different activity spectra between chlorooxirane and its homolog epichlorohydrin points out the necessity for a multifaceted study of carcinogens.V. V. SUMMARY Our laboratories have utilized strains of EL subtilis and Salmonella typhimurium to investigate the mutagenicity of vinyl chloride : tabolites and related compounds. The major findings reported in this tuscript are: 1) Confirmation of mutagenicity of chloroacetaldehyde i chlorooxirane. 2) Description of mutagenicity of additional potential metabolites of vinyl chloride, chloroacetaldehyde monomer hydrate, dimer hydrate, and trimer, as well as the mutagenic CMA 003727 STUDIES ON THE MUTAGENICITY OF VINYL CHLORIDE METABOLITES carcinogenic chlorooxirane homolog, epichlorohydrin. 3) Recombination repair is postulated to be the mechanism for correcting vinyl chloride metabolite elicited damage. 4) Chloroacetaldehyde affects the transformation activity of DNA only if cells are treated with the mutagen prior to the extraction of the DNA. In vitro the chemical had no effect. 5) Epichlorohydrin differs from vinyl chloride metabolites in mode of action. VI. ACKNOWLEDGEMENTS We wish to thank Mary A. Kinnaman for her extremely able technical assistance. We are grateful to Dr, Jerald Hoffman for malting available preliminary results and to Dr. B. N. Ames for providing the Salmonella tester strains. This work was supported by a grant from the B. F. Goodrich Company to the Cancer Center at the University of Louisville, School of Medicine. VII. LITERATURE CITED 1. Ames, B.N., Lee, F.D., and Durston, W.E. An Improved Bacterial Test System For Detection And Classification Of Mutagens And Carcinogens. Proc. Nat. Acad. Sci. U.S.A., 70: 782-786, 1973. 2. Barrio, J.R., Secrist, J.A., and Leonard, N.J. Fluorescent Adenosine And Cytidine Derivatives. Biochem. Biophys. Res. Comm., 46: 597-604, 1972. 3. Bartsch, H., Malaveille, C., and Montesano, R.M. Human, Rat, And Mouse Liver-Mediated Mutagenicity Of Vinyl Chloride In S. typhimurium Strains. Int. J. Cancer, 15: 429-437, 1975. 4. Creech, J.L., and Johnson, M.N. Angiosarcoma Of The Liver In The Manufacture Of Polyvinyl Chloride. J. Occup. Med., 16_: 150-151, 1974. 5. Ganesan, A.K., and Smith, K.C. Recovery Of Recombination Deficient Mutants Of Escherichia coli K-12 From Ultraviolet Irradiation. Cold Spring Harbor Symp. Quant. Biol., S3: 235-242, 1968. 6. Ganesan, A.T., and Lederberg, J. A Cell-Membrane Bound Fraction Of Bacterial DNA. Biochem. Biophys. Res. Comm., 1: 824-835, 1965. 7. Hefner, R.E., Watanabe, P.G., and Gehring, P.G. Preliminary Studies Of The Fate Of Inhaled Vinyl Chloride Monomer (VCM) In Rats. Ann. N. Y. Acad. Sci., 246: 135-148, 1975. 8. Jensen, R.A., and Haass, F.L. Analysis of Ultraviolet Light-Induced Mutagenesis By DNA Transformation In Bacillus subtilis. Proc. Nat. Acad. Sci. U.S.A., 50: 1109-1116, 1963. 9. Kada, T., Tutikawa, K., and Sadaie, Y. In Vitro And Host-Mediated "Rec-Assay" Procedures For Screening Chemical Mutagens; And Phloxino, A Mutagenic Red Dye Detected. Mutation Rea., 1(3: 165-174, 1972. CMA 003728 STUD1-5 transformation activity of DNA only if cells are treated trier the mutagen prior to the extraction of the DNA. In vitro the chemical had no effect. 5) Epichlorohydrin differs from vinyl chloride metabolites in mode of action. ACKNOWLEDGEMENTS We trish to thank Mary A. Kirxaman for her extremely able technical assistance. We are grateful to Dr. Jerald Hoffman for making available preliminary results and to Dr. 3. N. Ames for providing the Salmonella taster strains. This vrsrl v.-as supported by a grant frsm tha'B. 7. Goodrich Company to the Cancer Center at the university of Louisville, VII. LITERATURE CITED 1. Ames, B.N., Lee, F.D., and Durston, W.E, An Improved Bacterial Test System For Detection And Classification Of Mutagens And Carcinogens. Proc. Nat. Acad. Sci. U.S.A., 70: 732-786, 1973. 2. Barrio, J.R., Secrist, J.A., and Leonard, N.J. Fluorescent Adenosine And Cytidine Derivatives. Biochem. Biophys. Res. Comm., 46: 597-604, 1972. 3* Bartsch, H., Malaveille, C., and Montesano, R.M. Human, Rat, And House Liver-Mediated Mutagenicity Of Vinyl Chloride In S. typhimurium Strains. Int. J. Cancer, 15: 429-437, 1975. 4 Creech, J.L., and Johnson, M.N. Angiosarcoma Of The Liver In The Manufacture Of Polyvinyl Chloride. J. Qccup. Med., 16: 150-151, 1974. 5. Ganesan, A.K., and Smith, K.C. Recovery Of Recombination Deficient Mutants Of Escherichia coli K-12 From Ultraviolet Irradiation. Cold Spring Harbor Syrop. Quant. Biol., 21} 235-242, 1968. 6. Ganesan, A.T., and Lederberg, J. A Cell-Membrane Bound Fraction Of Bacterial DNA. Biochem. Biophys. Res. Comm., 18: 824-835, 1965. 7. Hefner, R.E., Watanabe, P.G., and Gehring, P.G. Preliminary Studies Of The Fate Of Inhaled Vinyl Chloride Monomer (VCM) In Rats. Ann. N. Y. Acad. Sci., 246: 135-148, 1975. 8. Jensen, R.A., and Haass, F.L. Analysis of Ultraviolet Light-Induced Mutagenesis By DNA Transformation In Bacillus subtilis. Proc. Nat. Acad. Sci. U.S.A., 50: 1109-1116, 1963. 9. Kada, T., Tutikawa, K., and Sadaie, Y. In Vitro And Host-Mediated "Rec-Assay" Procedures For Screening Chemical Mutagens; And Phloxine, A Mutagenic Red Dye Detected. Mutation Res., 16: 165-174, 1972. CMA 003729 Laumbach, A. D., Lee, S., Wong, J., and Streips, U. N. 10. Laumbach, A. D., and Felkner, I. C. Formation Of A 4-Nitroquinoline-l-Oxide Complex V/ith DMA In Normal And Repair-Deficient Strains Of Bacillus subtilis. Mutation Res., 15: 233-245, 1972. 11. Lee, C. H., and Wetmur, J. G. Physical Studies Of Chloroacetaldehyde Labeled Fluorescent DNA. Biochem. Biophys, Res. Commun., 50: 879-885, 1973. 12. Lee, F. I,, and Harry, D. S. Angiosarcoma Of The Liver In A Vinyl Chloride Worker. Lancet, 1: 1316-1318, 1974. 13. Lehmann, A. R. Postreplication Repair Of DNA In Mammalian Cells. Life Sci., 15: 2005-2016, 1974. 14. Malaveille, C. H., Bartsch, H., Montesano, R., Barbin, A., Camus, A. M., Croizy, A., and Jacquignon, P. Mutagenicity Of Vinyl Chloride, Chloroethylene Oxide, Chloroacetaldehyde And Chloroethanol. Biochem. Biophys. Res. Commun., 63: 363-370, 1975. 15. Maltoni, 0., and Lefemine, G. Carcinogenicity Bioassays Of Vinyl Chloride. Environm. Res., Tj. 387-405, 1974. 16. McCann, J., Simmon, V., Streitv/ieser, D., and Ames, B. N. Mutagenicity Of Chloroacetaldehyde, A Possible Metabolic Product Of 1,2-Dichloroethane (Ethylene Dichloride), Chloroethanol (Ethylene Chlorohydrin), Vinyl Chloride, And Cyclophosphamide. Proc. Nat. Acad. Sci., U.S.A., 72: 3190-3193, 1975. 17. Okubo, S., and Romig, W. R. Impaired Transformability Of Bacillus subtilis Mutant Sensitive To Mitomycin C And Ultraviolet Radiation. J.' 'Mol."Biol., 15: 440-454, 1966. 18. Olsen, W. L., Heidrich, H. G., Hannig, K., and Hofshneider, P. H. Deoxyribonucleic Acid-Envelope Complexes Isolated From Escherichia coli By Free-Flow Electrophoresis: Biochemical And Electron Microscope Characterization. J. Bacteriol., 118: 646-653, 1974. 19. Rannug, U., Johansson, A., Ramel, C., and Wachtmeister, C. A. The Mutagenicity Of Vinyl Chloride After Metabolic Activation, `'-{BIO, 3: 194-197, 1974. 20. Richards, G. Modifications Of The Diphenylamine Reaction Giving Increased Sensitivity And Simplicity In The Estimation Of DNA. Anal. Biochem., 57_: 369-376, 1974. 21. Strauss, B., and Okubo, S. Protein Synthesis And The Induction Of Mutations In Escherichia coli By Alkylating Agents. J. Bacteriol., 79: 464-473, 1950l 22. Strauss, B., Reiter, H., and Searashi, T. Recovery From Ultra violet And Alkylating Agent-Induced Damage In Bacillus subtilis. Rad. Res. Supp., 6_: 201-211, 1966. 23. Streips, U. N., and Young, F. E. Transformation In Bacillus subtilis Using Excreted DNA. Molec. Gen. Genetics, 133: 47~55, 1974. CMA 003730 STUDIES ON THE MUTAGENICITY OF VINYL CHLORIDE METABOLITES 24. Sueoka, N., and Quinn, V/. Membrane Attachment Of The Chromosome Replication Origin In Bacillus subtilis. Cold Spring Harbor Symp. Quant. Biol., 33: 695-705, 1968. 25. Van Duuren, B. L. On The Possible Mechanism Of Carcinogenic Action Of Vinyl Chloride. Ann. N. Y. Acad. Sci., 246: 258-267, 1975. 26. Viola, P. L., Bigotti, A., and Caputo, A. Oncogenic Response Of Rat Skin, Lungs And Bones To Vinyl Chloride. Cancer Res., 31.: 516522, 1971. 27. Vogel, H. J., and Bonner, D. M. Acetylomithinase Of Escherichia coll: Partial Purification And Some Properties. J. Biol. Chen., 2TST 97-106, 1956. 28. Yamagudin, K., and Yoshikawa, H. Association Of The Replication Terminus Of The Bacillus subtilis Chromosome To The Cell Membrane. J. Bacteriol., 124: 1030-1083, 1975. 29. Young, F. E., and Wilson, G. A. Bacillus subtilis. In: Handbook Of Genetics. Ed.: Robert C. King, Plenum Press, New York, 1_: 69114, 1974. CMA 003731 25 Van Duuren, B. L. On Th Possible Mechanism Of Carcinogenic . Of Vinyl Chloride Ajin # M. Y. Acad. Sci., 216: 253-267, 1975 Rat Skin, Lungs And Bcnes To Vinyl Chloride. Cancer Res., 31: 516522, 1971. 27. Vogel, H. J. , and Bonner, D. M. Acetylomithinase Of Escherichia coli: Partial Purification And Sene Properties. J. Biol. Chen., 213: 97-106, 1956. 23. Yaraagudin, K., and Yoshikawa, H. Association Cf The Replication Terninus Cf The Bacillus suetill3 Chrcnoscne To The Cell Membrane. J. Bacteriol., 124: 1030-1033, 1975. 29. Young, F. E., and Wilson, G. A. Bacillus subtilis. In: Handbook Of Genetics. Ed.. : Robert C. F-ing, Plenum Press, New York, 1: 69114, 1974. CAiA 03733 Laumbach, A. D., Lee, S., Wong, J., and Streips, U. N. Table I Bacterial Strains Bacillus subtilis Genotype Origin and Comments RUB 783 BR 151 BUL 709 BUL 714 Hcr-9 (JB01-200) MC-1 FB-13 16 SWT purB6, leu-8, hisAl, metBlO trpC2, lys-3, metBlO ura-1, hisAl, leu-8, metBlO cysA, hisAl, leu-8, :metBlO trpC2 trpC2, recB2 trpC2 prototroph U. Streips B. Reilly This laboratory This laboratory S. Okubo and W. Romig, her- S. Okubo and W. Romig, recC. Hadden, uvrA. Laumbach and I. Felkner Salmonella typhimurium imitations in Strains His- LPS DNA Repair R Factor Mutation Detected TA1535 TA100 TA1537 TA1538 TA98 hisB46 rfa hisB46 rfa hisC3076 rfa hisD3052 rfa hisD3052 rfa uvrB uvrB uvrB uvrB uvrB pKMIOl pKMIOl base-pair substitution base-pair substitution frameshift frameshift frameshift CMA 003733 STUDIES ON THE MUTAGENICITY OF VINYL CHLORIDE METABOLITES Table II Mutagenic Activity Assayed by Bacterial Test Systems Compounds Indirect Screen B. subtilis "Repair-Ass ay" Direct Testa S. typhimurium Strain TA100 Acetaldehyde Chloroacetic Acid Chloroethanol Vinylidene Chloride Vinyl Chloride Chlorooxirane Chloroacetaldehyde (monomer) Chloroacetaldehyde (monomer-dimer hydrates) Chloroacetaldehyde (dimer hydrate) Chloroacetaldehyde (trimer) Epichlorohydrin NRb NR NR NR NR +c +++e ++ + + NR NR NR NR NR NR ++d ++ + ++ + + + Experiments performed in absence of liver homogenatemediated activation. ^NR no reaction detected c + Reactive d++ Moderately reactive e+++ Very reactive CMA 003734 )IES ON THE .5-Sv i>j'3Cc.T-3 Compounds ^ f*V * C ^ B. subtilis "Repair-Assay" Direco .esc S. typhimurium Strain TA100 Acetaldehyde C n.1 c -C 6*t 1 o Ac 1d Chloroethar.ol Vinylidene Chloride Vinyl Chloride Chlorooxirane Chloroacetaldehyde (monomer) Chloroacetaldehyde (monomer-dimer hydrates) Chloroacetaldehyde (dimer hydrate) Chloroacetaldehyde (trimer) Epichlorohydrin NRb >;p MR NR NR +0 +++e ++ + + NR NR \"2 MR NR MR ++d ++ + ++ + + + aExperiments performed in absence of liver homogenatemediated activation. no reaction detected G + Reactive ^++ Moderately reactive e+++ Very reactive cMA 003735 Laumbach, A. D ., Lee, S ., Wong, J . , and S tre ip s CMA 0 0 3 7 3 6 Table III "Repair-Assay" with Bacillus subtilis Strains Compounds Molar Concentration Growth Inhibition in Millimetersa 168WT MC-1 Hcr-9 FB-13 (hcr+, rec + ) (hcr+, rec-) (her-, rec+) ( uvr+, rec+) Chloroacetaldehyde (monomer) 0.10 2.0 28.0 4.0 3.0 Ohioroacetaldehyde (monomer-dimer hydrate) 0.115 NIb 23.0 NI NI Chloroacetaldehyde 0.097 2.0 10.0 2.0 2.0 (dimer hydrate) Chloroacetaldehyde (trimer) 0.096 7.0 15.0 6.0 7.0 Chlorooxirane 0.26 NI 10.0 NI NI Epichlorohydrin 0.997 NI NI NI NI Epichlorohydrin (plus liver homogenate)0 0.997 NI 3.0 NI NI aAverage inhibition calculated from multiple experiments. bNo inhibition detected. 9,000 x g supernatant (S-9) + NADPH generating system. STUDIES ON THE MUTAGENICITY OF VINYL CHLORIDE METABOLITES 003737 Table IV Quantitative Mutagenicity Assay by Salmonella TA200 Reversion Compound Concentration In Soft Agar Layer mM/Platea Chloroacetaldehyde (monomer) Chloroacet aldehyde {monomer-dimer hydrate) Chloroacetaldehyde (dimer hydrate) Chloroacetaldehyde (trimer) Epichlorohydrin 0.0004 0.054 0.490 0.240 4.746 highest effective non-toxic concentration for reversion. ^Spontaneous background revertants subtracted. Average Number Revertants/Plate^ 265 977 311 159 2856 u Table IV Quantitative Mutagenicity Assay by Salmonella TA100 Reversion oo co Concentration in co o o iM1M\' Compound Soft Agar Layer mM/Platea Average Number Re vert ants/Plate^ U ri \! Chloroacetoldehyde 0.0004 265 i'j (monomer) UO3 ni l>? Chloroacetaldehyde (monomer-dimer hydrate) 0.054 977 r -> .1 Chioroac c t ald ehyde (dimer hydrate) 0.490 311 Chloroacetaldehyde 0.240 ; (trimer) 159 ( Epiclilorohydrin lt 4.746 2856 !! highest effective non-toxic concentration for reversion. I ^Spontaneous background revertants subtracted. iI i1 i<0* Laumbach, A. D ., Lee, S ., Wong, J . , and S tr e ip s , 003739 Table V Effect of Chioroacetaldehyde and Epichlorohydrin of Transforming DNA In vitro Recipient Strains Relative Transformation Efficiency3 rnetBlO leu-Q cysA hisAl ura-1 trpC2 lys-8 purB6 U Epichlorohydrin treated DNA BUL 714 .92 .97 .97 1.16 RUB 783 .91 .60 .98 .77 BUL 709 .99 .95 1.02 .85 BR 151 1.48 1.07 .62 Chloroacetaldehyde treated DNA BUL 714 1.43 .92 .55 '.91 RUB 783 .93 1.45 .75 .89 BUL 709 BR 151 .86 1.33 .90 .75 NDb .77 Relative transformation efficiency calculated: number of transformants with treated DNA b^ot determined number of transformants with untreated DNA cConditions for competence and transformation as described in Materials and Methods. Table VI EFFECT OF CHLOROACETALDEHYDE AND EPICHLOROHYDRIN ON TRANSFORMING DNA IN VIVO STUDIES ON THE MUTAGENICITY OF VINYL CHLORIDE METABOLITES 003740 Recipient Strains13 Relative Transformation Efficiency^ metBlO leu-8 cysA hisAl ura-1 trpC2 lys-3 purBl6 Chloro ac e t aldehyd e in vivo treated DNA BUL 714 RUB 783 BUL 709 BR 151 Epichlorohydrin in vivo treated DNA .54 .09 .08 .36 .33 .10 .35 .53 .07 .32 .34 .37 .13 .11 .10 BUL 714 RUB 783 BUL 709 BR 151 .55 .17 .25 .46 .52 .15 .38 .59 .15 .50 .36 .37 .11 .19 NDC Relative transformation efficiency calculated: number transformants with treated DNA number transformants vrith untreated DNA ^Conditions for competence and transformation as described in Materials and Methods. cNot determined. Table VI EFFECT OF CHLOROACETALDEHYDE AND EPICHLOROHYDRIN ON TRANSFORMING DNA IN VIVO Recipient Strains*5 Relative Transformation Efficiency me t,B10 leu-8 cysA hisAl ura-1 trpC2 lys-3 purBl6 wom o o t\>r 1tii Chloroacetaldehyde in vivo treated DMA f? L'J U iint. ;ji BUL 714 RUB 783 .54 .09 .08 .36 33 .10 .35 .10 .i BUL 709 .53 .07 .32 .34 I- 4 BR 151 37 .13 .11 Epichlorohydrin Ot-t in vivo treated DNA \oA -t; BUL 714 .55 .17 .25 .46 RUB 783 .52 .15 .38 aM BUL 709 .59 .15 .50 .36 NDC *M0 BR 151 p-i .37 .11 .19 u o(- Relative transformation efficiency calculated: number transformants with treated DNA number transformants with untreated DNA ^Conditions for competence and transformation as described in Materials and Methods. cNot determined. VINYL CHLORIDE MUTAGENICITY AND CARCINOGENICITY VIA THE METABOLITES CHLOROOXIRANE AND CHLOROACETALDEHYDE MONOMER HYDRATE. Jim D. Elmore and John L. Wong* Department of Chemistry, University of Louisville Andrew D. Laumbach and Uldis N. Streips* Department of Microbiology, University of Louisville Louisville, Kentucky, USA 40208 . SUMMARY Mutagenicity tester strains of Bacillus and Salmonella were used to assay vinyl chloride in nutrient broth at a practical concentration level. Also screened without exogenous activation were seven potential metabolites of vinyl chloride in their pure forms as well as the related epichlorohydrin. Chlorooxirane, chloroacetaldehyde, chloroacetaldehyde monomer hydrate, chloroacetaldehyde dimer hydrate, chloroacetaldehyde trimer, and epichlorohydrin produced significant mutagenic activity in Salmonella typhimurium strains sensitive to base-pair mutation. A recombination repair deficient strain of Bacillus subtil is was inhibited in growth by these compounds, whereas excision repair deficient and wild type strains of Bacillus subtil is were relatively unaffected. On the basis of these assays a working hypothesis for the vinyl chloride carcinogenesis mechanism is proposed which involves chlorooxirane and chloroacetaldehyde monomer hydrate as the ultimate carcino genic metabolites of vinyl chloride. CJVZA 03742 L INTRODUCTION The carcinogenic potential of vinyl chloride monomer 1 was initially 'V established by Viola et [1] and Maltoni et al. [2] with inhalation experi ments using laboratory animals. Detection of angiosarcoma in polyvinyl chloride workers suggested a causal relationship between industrial exposure to vinyl chloride and the development of pathological conditions in humans. This contention was supported by epidemiological data which revealed an association between exposure to 1 and the onset of hepatic abnormalities including angio- ' A/ sarcoma [3,4]. A report by Hefner et_ al_. [ 5] on the metabolic fate of 1 in rats indicated that 67% of the vinyl chloride inhaled by the rats was metabolized and excreted in the urine. The metabolic products identified were N-acetyl-S- (2-hydroxyethyl)cysteine and thiodiglycolic acid [5,6] which are sulfhydryl conjugates of chloroethanol f2\j and chloroacetic acid i3\j respectively. Chloro-' r ' oxirane 4 and chloroacetaldehyde 5 v/ere speculated to be the carcinogenic forms. We report herein the mutagenicity and carcinogenic potential of these i compounds in their pure forms. Several reports [7,8,9] have appeared recently using the Salmonella tester strains to test vinyl chloride and several of its supposed metabolic derivatives. Vinyl chloride and chloroethanol were found to be mutagenic after activation by liver homogenates [9]. Direct mutagenicity of vinyl chloride was also reported by McCann ejt^ aj_. [7] and Bartsch et_ah [10] at 202 v/v in. air (200,000 ppm). Since the solubility of vinyl chloride in water at 25C and 1 atm has been determined to be 7.79 x 10"^ mole fraction [11] or 2,900 ppm, we have conducted further testing at this concentration level to secure a practical CMA 003743 j dose-response comparison with its proximate metabolites. Regarding the latter, the exact chemical forms of the proximate metabolites previously tested are often questionable. Chloroacetaldehyde, like formaldehyde [12], dichloroacetaldehyde [13], and chloral [14], can exist in combinations of four forms depending on the history of sample preparations: the monomer 5, the 'V monomer hydrate *6\l, the dimer hydrate <7\r, and the trimer <8\,. McCann -e--t- --al. [7] used vacuum distilled chloroacetaldehyde without a follow-up analysis of its content. This distillate may have consisted of chloroacetaldehyde monomer 5 and its cyclic trimer 8'V if water was totally absent, or it may have been a mixture of chloroacetaldehyde hydrates 6 and 7 in an aqueous medium. Bartsch et al_. El0] tested a commercial aqueous chloroacetaldehyde solution which, according to our analysis reported herein, had an acidic pH and approximately equal concentrations of the two hydrates 6 and 7. This solution was also contaminated by ethanol to the extent of 10%. We have therefore conducted individual assays of pure compounds, or assays of a known mixture of the specific forms of chloroacetaldehyde. Furthermore, the mutagenicity observed for chlorooxirane 4 [9] may be attributed to a chloroacetaldehyde hydrate rather than the chlorooxirare integrity. Under the 37C aqueous testing conditions reported, chlorooxirane decomposed with a half life of 1.6 min [10] to chloroacetaldehyde. For this reason we have also screened epichlorohydrin 9 which is a stable chloro-epoxide homolog of 4 as a comparative assay to interpret the observations of the activity of chlorooxirane. This investigation used the above-mentioned compounds 1 - 9 in mutagen assays without exogenous enzyme activation. A preliminary screen was performed using DNA repair-deficient mutants of Bacillus subtil is. This was followed by quantitative testing of the compounds for mutagenicity CMA 003744 forms depending on the history of sample preparations: the monomer 5, the % monomer hydrate 6'V, the dimer hydrate '7V,, and the trimer 8. McCann -e--t---a--l. [7] used vacuum distilled chloroacetaldehyde without a follow-up analysis of its content. This distillate may have consisted of chloroacetaldehyde monomer 5 r - - tr i'iiS ^ *r- a y it, or it may nave oe=r. mixture of chloroacetaldehyde hydrates 5 and 7 in an aqueous medium. Sartscn et aU hol tested a commercial aqueous chloroacetaldehyde solution which, according to our analysis reported herein, had an acidic pH and approximately equal concentrations of the two hydrates %6 and '7V. This solution was also contaminated by ethanol to the extent of 102. We have therefore conducted individual assays of pure compounds, or assay,-, of a known mixture of the specific forms of chloroacetaldehyde. Furthermore, the mutagenicity observed for cnlorooxirane 4 [9] may be attributed to a chloroacetaldehyde hydrate rather than the chiorooxirare integrity. Under the 37C aqueous testing conditions reported, chiorooxirane decomposed with a half life of 1.6 min [10] to chloroacetaldehyde. For this reason we have also screened epichlorohydrin 9 which is a stable chloro-epoxide homolog of 4 as a comparative assay to interpret the observations of the activity of chlorooxirane. This investigation used the above-mentioned compounds 1^ - 9 in mutagen assays without exogenous enzyme activation. A preliminary screen was performed using DNA repair-deficient mutants of Bacillus subtil is. This was followed by quantitative testing of the compounds for mutagenicity Cma 003745 4 with Salmonella typhimurium LT-2 strains [15], Thus, a combination of these two screening procedures have led to information on the mutagenicity and carcinogenic potential of these compounds as well as their chemical mode of action. MATERIALS AND METHODS The bacterial strains used in the bioassays are shown in Table I. The Salmonella tester strains were designed to detect chemical carcinogens as mutagens [15]. The recombination and DNA repair-deficient Bacillus subtil is strains were used in tiie repair assays as an indirect test for mutagenicity [16,17], Nutrient broth (Difco) and nutrient broth plus 0.5% NaCl was used for growth of stock cultures of Bacillus and Salmonella strains respectively. Nutrient agar (Difco) served as a solid medium for the growth of Bacillus strains in "repair-assays". The pour plates used with Salmonella strains consisted of molten (45C) soft agar which contained 0.6% agar, 0.6% NaCl, 0.5mM biotin, and 0.5 mM histidine. The minimal agar plate was composed of Vogel-Bonner E medium [18], 1.5% agar, and 2% glucose. Vinyl chloride gas was obtained from Matheson Scientific; aqueous chloroacetaldehyde (45% by wt.) from ICN Pharmaceuticals; epichlorohydrin from Matheson Coleman and Bell; and other chemicals from Aldrich Chemical Co. Mutagenicity Assays Salmonella - Vinyl chloride 1 was tested by the method of Ames [7]. A mixture of 0.1 ml of 1 in broth and 2 ml of top agar was added to 0.1 ml of cell culture. The solutions were then mixed and poured immediately onto the surface of a minimal medium plate. After incubation for 48 hrs at 25"C, colonies were counted and recorded. For compounds 2 - 9, sample solutions of known concentrations CMA 003746 were prepared in dimethyl sulfoxide (DMSO). A 0.1 ml aliquot of the sample solution was admixed with 0.9 ml of the tester strain culture. Then, a 0.1 ml sample of this mixture was added to 2 ml of molten soft agar and applied to the surface of a minimal agar plate. Control plates for detection of the spontaneous reversion rates were prepared for each tester strain by omitting only the compounds tested. Pour plates were incubated for 48 hr at Z7C before revertant colonies to prototrophy were counted. For positive mutagenesis control, plates containing the mutagen 4-nitroquinoline-N-oxide ./nre used. Bacillus - The "repair-assay" procedure was a modification of the "recassay" procedure of Kada et al. [19]. They were grown overnight in nutrient broth then diluted 10 fold in phosphate buffer (pH 7.0). Strains were streaked with pipettes onto nutrient agar plates. Filter paper discs (6 mm) saturated with test solution were placed upon the bacteria streaks. Following incubation for 24 hr at 37C, growing bacteria were visible except in the inhibition zone The lethality and mutagenic potential of compounds were assessed by comparison of inhibition zones between the 168 wild type strain and the DNA repair-deficient strain and the DNA repair-deficient strains. The control used was 4-nitroquinol ineN-oxide. Survival assays for 6 and 7 (cf.. Fig. 3.) were made in MY-1 broth [17] solutions. Cultures were grown in tryptose blood agar base for 16 hrs, inoculated into MY-1 broth, and viable cell counts were done on TBAB agar plates. Compound Synthesis and Purification Chiorooxirane 4 prepared by the method of Walling and Frederick [20] was in higher purity (95% pure) than that by molecular chlorination [21] (50% pure). Thus, t-butyl hypochlorite and ethylene oxide at -10C with 200 watt tungsten lamp irradiation yielded chlorooxirane 4; glpc (gas liquid phase chromatography) CMA 003747 = 1.2 min at 508C, infrared absorption ( v cm ^} 900, 1250, 1320, and 1710 as described previously [21], and PMR as shown in Table II. Derivatization of 4 with an excess of acidic 2,4-dinitrophenylhydrazine solution gave glyoxal-bis-dinitrophenylhydrazone, mp 317-318C (317*C reported[21]). Chloroacetaldehyde monomer 5 was obtained in the purest form by cracking the chloroactaldehyde trimer 8 at 95C and distilling it into dry DMS0; glpc tjj * 2.25 min at 100C and PMR as shown in Table II. Chloroacetaldehyde dimer hydrate 7 - A solution containing 49.5 ml of 38% hydrochloric acid, 30 ml of the 45% aqueous chloroacetaldehyde solution, and 55.5 ml of water v/as distilled over a 9 inch Vigreaux fractionating column. The fraction (1/5 of the initial volume) collected from 87-100C was redistilled. This second distillate at 83-92C crystallized after 3 days at -15C. Upon sublimation of 60C and 1 atm, white crystals of 7 were obtained; mp 55C and PMR as shown in Table II. Chloroacetaldehyde trimer 8 - Concentrated sulphuric acid (7.5 ml) was added to the 45% aqueous chloroacetaldehyde solution (5 ml) with vigorous stirring and external cooling (-5C). The crystalline precipitate was filtered after standing overnight at -15C, washed with 5 ml of cold 20% aqueous methanol, and recrystallized 5 times from cold methanol; mp 87-888C, corresponding to that reported by Natterer [13], and pMR as shown in Table II. Quantitation of vinyl chloride in nutrient broth - The concentration of vinyl chloride 1 in the broth solution was determined by an extraction method in conjunction with glpc. This method involved (1) establishing the CMA 003748 7 linearity of the response of 1 on glpc, (2) constructing a standard curve of vinyl chloride weight vs_. peak area, and (3) extracting the broth with methylene chloride followed by glpc determination. (1) Linearity of "response - A standard solution of '1Xf was prepared by condensinq it (bp -13.4C) at -78C onto a known weight of methylene chloride in a 1 ml volumetric flask fitted with a serum cap. The condensed vinyl chloride was determined by weighing. A typical solution thus prepared was 0.2877 M and was subjected to glpc analysis by varying injection sizes from 1-9 yl. The correlation of vinyl chloride weight and peak area was made by a least square computer routine: slope = 0.877, with an index of correlation of 0.972 (ideal 1.00) up to 6 yl (0.1078 mg) of injection. (2) A standard linear curve using the above technique was established for 0.01-0.10 mg of vinyl chloride. (3) Extraction of broth Vinyl chloride v/as allowed to bubble through the nutrient broth for 30 min at 258C. A 1.0 ml broth sample was then extracted with 4 x 2 ml of methylene chloride, the extracts were combined and then made up to 10 ml in a volumetric flask with methylene chloride. Glpc analysis of this solution and application of the standard curve showed that there was 7.0 mg of 1 in the 10 ml solution, or the concentration of 1 in the broth was 0.0107 M. Repeated determination showed it to be 0.0105 M. High Pressure Liquid Chromatographic (HPLC) Analysis of the Commercial 45% Aqueous Chloroacetaldehyde - Reverse phase HPLC on the commercial 45% chioroacetaldehyde solution (7 M, pH 2.6) resolved it into two components: tfJ = 6.3 min and tD2 = 8.8 min in a ratio of 40:60. - The ratios of the two peaks on the chromatogram changed as the solution pH was varied by addition of 1 N MaOH at room temperature: 43:57 (pH 3.7), 44:56 (pH 5.0), 48:53 (pH 7.6), 003^49 \ 0 ' r.l solution was admixed witn 0.3 mi of cne Lester strain culture. Then, a J zzz ^ * u ..........`~'i <i5 - r p.-2* Ccn^i'c* piatss for CctcCG*. on of the spontaneous reversion rates were prepared for each tester strain by omitting only the compounds tested. Pour plates were incubated for 48 hr at 37C before revertant colonies to prototrophy were counted. For positive mutagenesis control, plates containing the mutagen 4-nitroquinoline-N-oxide rre used. aoil 1 us - The "repair-assay" procedure was a modification of the "recassay" procedure of Kada et_ al_. [19]. They were grov/n overnight in nutrient broth then diluted 10 fold in phosphate buffer (pH 7.0). Strains were streaked with pipettes onto nutrient agar plates. Filter paper discs (6 mm) saturated with test solution were placed upon the bacteria streaks. Following incubation for 24 hr at 37C, growing bacteria were visible except in the inhibition zone. The lethality and mutagenic potential of compounds were assessed by comparison of inhibition zones between the 168 wild type strain and the DNA repair-deficient strain and the DNA repair-deficient strains. The control used was 4-nitroquinoline N-oxide. Survival assays for 6 and 7 (cf.. Fig. 3.) were made in MY-1 broth [17] solutions. Cultures were grown in tryptose blood agar base for 16 hrs, inoculated into MY-1 broth, and viable cell counts were done on TBAB agar plates. Compound Synthesis and Purification Chlorooxirane 4 prepared by the method of Walling and Frederick [20] was in higher purity (95% pure) than that by molecular chlorination [21] (50% pure). Thus, t-butyl hypochlorite and ethylene oxide at -10C with 200 watt tungsten lamp irradiation yielded chlorooxirane 4; glpc (gas liquid phase chromatography) *\# CMA 003750 {CANCER RESEARCH 41, 419-424, February 1981) 000^5472/81/0041-0000SO2 00 Tissue and Urinary Glycosaminoglycan Patterns Associated with a Fast, an Intermediate, and a Slow-growing Morris Hepatoma*1 Charles E. Kupchella,2 E. Elaine Drake, Jeffrey Kennedy, Kevin L. Curran, Raya Warick, and H. P. Morris Cance/1 Center, University ot Louisville. Lou,sville. Kentucky 40201 {C E K.E E D.J K.K t C n W l and the Department of Biochemistry. Cancer Research Unit. College of Medicine. Howard University. Washington o C IH R K! ] ABSTRACT MATERIALS AND METHODS The purpose of this investigation was to evaluate the glycosaminoglycans (GAG's) in different behavioral-histological types of i.m.-transplanted hepatomas and in the liver and urine of animals bearing these tumors. Groups of 10 Buffalo rats carrying fast-growing (7777). intermediate (5l23tc). and slowgrowing (961SA) Morris hepatomas were studied as the tumors reached 3 cm. Urinary and tissue GAG'S were isolated by proteolysis, separated as cetylpyridinium complexes, and measured as uronic acid. The GAG's were further purified using anion-exchange chromatography and characterized with mucopolysaccharidases. Tissue GAG's were also evaluated histochemically using Alcian blue staining and mucopolysac charidases. Tissue from fast-growing, intermediate, and slowgrowing tumors exhibited greater GAG levels than did normal liver in the hyaluronic acid (0.4 m NaCI-soluble) fraction and in the chondroitin sulfate-heparan sulfate (1.2 m NaCI-soluble) fraction. The livers of tumor-bearing animals exhibited GAG levels similar to those of normal liver. Increased urinary GAG excretion was appreciated in animals bearing Tumors 5123tc and 9618A but not in those bearing Tumor 7777. INTRODUCTION An increasing number of reports cite the presence of com paratively high levels of GAG's3 in both animal tumors (4, 7, 18, 22) and human tumors (5. 12, 16), There also have been reports citing qualitatively and quantitatively abnormal urinary GAG excretion in association with malignant tumors (6,10,11, 23-25). Although there have been attempts to establish any functional relationship between tumor GAG's and tumor cell properties (23, 25-27, 30, 31), the significance of elevated GAG'S in malignant tumors remains obscure. In view of the possibility that GAG's play an important role in the expression of one or more malignant cell properties, our purpose here was to evaluate the GAG patterns associated with transplantable hepatomas exhibiting different growth rates and metastatic properties. Because it has been suggested that tumor GAG may be contributed by normal host tissue in re sponse to the presence of hepatic tumor (7), a secondary purpose was to evaluate the influence, if any, of "remote" hepatomas on the GAG's of the host liver; a third purpose was to evaluate the urinary GAG patterns in hepatoma-bearing animals. * Supported by American Cancer Society Grant tN-i 11B. by a grant from the Manufacturing Chemists Association, and in part by USPHS Grants CA 10729 and CA 246201. 1 Present address: BWogidal Sciences, Murray State University. Murray, Ky. 42071. To whom requests for reprints should be addressed. * The abbreviation used is: GAG, Oiycosaminogtycan. Received April 11, 1960: accepted October 24, i960. Materials. Hyaluronic acid (umbilical cord) was purchased from Nutritional Biochemical Corp. (Cleveland, Ohio); chon droitin sulfate (whale and shark cartilage) and sodium heparin were purchased from Sigma Chemical Co, (St. Louis, Mo.). Authentic samples of heparin, chondroitin 4-sulfate, heparan sulfate, and hyaluronate were also kindly supplied by Dr. M. B. Matthews. University of Chicago. Bovine testicular hyaluronidase was purchased from ICN Pharmaceuticals (Cleveland, Ohio), Streptomyces hyaluronidase was obtained from Calbiochem (La Jolla. Calif.), and Proteus vulgaris chondroitinase ABC was purchased from Sigma. Experimental Design. Forty male Buffalo rats were shipped from Lab Supply Company (Indianapolis, Ind.) to Washington, D. C.. where 10 were inoculated bilaterally (thigh) with Tumor 7777, 10 were inoculated with Tumor 5l23tc, and 10 were inoculated with Tumor 9618A. These were shipped to Louisville with 10 controls. Throughout the study, animals were provided free access to food and water even when placed in metabolic cages for urine collections. Urine collections were made twice each week from animals bearing Tumor 7777 and once each week from animals bearing Tumors 5l23tc and 9618A. Collections were made alterna tively on one-half of the animals in each group, with 5 control animal collections made each time a collection was made from tumor-bearing animals. Tumors. Line 5l23tc is a tissue culture variant of a moder ately differentiated trabecular hepatocellular carcinoma in duced with dietary administration of AI-2-fluorenylphthalamic acid. Tumors were received and studied here in the 165th generation. Tumor line 7777 is a poorly differentiated hepatocellular carcinoma induced by dietary administration of N-2fluorenyiphthalamic acid. This line was studied in the 159th transplant generation. Tumor line 9618A is a well-differentiated hepatocellular car cinoma induced by 2-(4'-methyl)benzoylaminofluorene. This tumor was studied in the 13th generation. Characteristics of these 3 tumors observed in our laboratory and selected characteristics reported by Hruban etal, (13,14) are summarized in Table 1 (see also Fig. 1). Extraction and Purification of GAG's from Liver and Tumor Tissue. Dry defatted tissue was subjected to proteolysis, tri chloroacetic acid precipitation, and dialysis, and th GAG's were separated as cetylpyridinium chloride complexes into 0.03 m NaCI-soluble, 0.4 m NaCI-soluble, 1.2 m NaCI-soluble. and 2.1 m NaCI-soluble fractions as described by Schiller et al. (28) and measured as uronic acid (see Schiller ef al.) by the carbazole method of Bitter and Muir (1). FEBRUARY 1901 CMA 003751 419 7 linearity of the response - t - - / 'i \ - -^r.-^>"*jr*^^^ ** j O' 7 1 <*y i C i i C r 1 C 3 Vi " i ^ i * ^ /_J_' r'- -* '' c .' v -* > '* ^ \ ' methylene chloride fot' ;sd cy glpc determination, Li r.oaritv response - A standard solution of 1 was prepared by condensing it (bo -13.4C) at -78C onto a known weight of methylene chloride in a 1 ml volumetric flask fitted with a serum cap. The condensed vinyl chloride was determined by weighing. A typical solution thus prepared was 0.2377 M and was subjected to glpc analysis by varying injection sizes frcm 1-9 vl. The correlation of vinyl chloride weight and peak area was made by a least square computer routine: slope s 0.377, with an index of correlation of 0.372 (ideal 1.00) up to 6 U1 (0.1078 mg) of injection. (2) A standard linear curve using the above technique was established for 0.01-0.10 mg of vinyl chloride. (3) Extraction of broth - Vinyl chloride v/as allowed to bubble through the nutrient broth for 30 min at 258C. A 1.0 ml broth sample was then extracted with 4 x 2 ml of methylene chloride, the extracts were combined and then made up to 10 ml in a volumetric flask with methylene chloride. Glpc analysis of this solution and application of the standard curve showed that there was 7.0 mg of 1 in the 10 ml solution, or the concentration of 1 in the broth v/as 0.0107 M. Repeated determination showed it to be 0.0105 M. High Pressure Liquid Chromatographic (HPLC) Analysis of the Commercial 45% Aqueous Chloroacetaldehyde - Reverse phase HPLC on the commercial 45% chloroacetaldehyde solution (7 M, pH 2.6) resolved it into two components: t^l = 6.3 min and t^2 = 8.8 min in a ratio of 40:60. The ratios of the two peaks on the chromatogram changed as the solution pH v/as varied by addition of 1 N NaOH at room temperature: 43:57 (pH 3.7), 44:56 (pH 5.0), 48:53 (pH 7.6), CMA 003752 and 56:44 (pH 8.2). The first eluted component at t^l = 6.3 min increased while the second at tD2 = 8.8 min decreased at raised pH's, This indicated a retrograde aldol condensation type hydrolysis. The first peak was assigned to the monomer hydrate 6 and the second to the dimer hydrate 7. When the 45% solution was diluted to 0.44 M at pH 3.6, refluxed for 1 hr, cooled, and chromatographed, the ratio of t^l:t^2 became 33:67. This suggests an acidcatalyzed condensation of 6 to form the dimer hydrate 7. When these two components were analyzed by glpc, both emerged at the same retention time (t~ = 2.8 min at 60C) as that of the monomer 5. Dehydration of 6 ana 7 must have occurred under the glpc conditions thereby reverting them to the monomer form. As shown in Table II, the PMR spectra of 6 and 7 are resolved from one another. Thus, the PMR spectrum of the 45% commercial solution also revealed the presence of both hydrates 6 and 7 with approximately the same integrals. Instrumentation High Pressure Liquid Chromatography (HPLC) - A Waters Associates model 600 pump combination (dual) with a model 440 differential 254 nm ultraviolet detector were used for the analysis of aqueous chloroacetaldehyde solutions. A Porasil Bondapak yC-|g4 mm x 30 cm column was used with a 80:20 v/v 0.1 N NH4H2P04-Me0H isocratic eluant (pH 4.9) at 1 ml/min. These two components were also resolved on a Reeve Angel Partisil 10 0DS 4.6 mm x 25 cm column using the same eluant. CMA 003753 9 Gas Liquid Phase Chromatography (GLPC) - Analysis of vinyl chloride 1, chlorooxirane 4, and chloroacetaldehyde 5 were performed on a Carle model 9500 flame ionization gas chromatograph, A 10% SE-30 on Anakron 60/70 packed column was used at 30C for vinyl chloride determination. A 20% Carbowax on chromosorb W column at 50C and 40 ml/rnin of Helium was used for analysis of 4 and 5 unless specified otherwise. Both were 0,125 inch x 5 feet stainless steel columns. Proton Magnetic Resonance (PMR) - Spectra were obtained using a Varian A-60 A and a Perkin Elmer R-12 spectrometer. Solutions of D2O and DMS0~dg were used with 3-(trimethylsilylJpropanesulfonic acid sodium salt as internal reference. Tetramethylsilane was used as a reference in CDCl^ and CCl^. Probe temperature was 38*0. RESULTS AND DISCUSSION Mutagenicity assays with Bacillus and Salmonella for compounds 1-9 are summarized in Table III. They are grouped into three categories in subsequent discussion. Further testing data are included in Tables IV-VI and Figures 2-4. Nonmutagenicity of vinyl chloride, chloroethanol, and chloroacetic acid Only high concentrations of vinyl chloride (20% v/v in air) have produced mutagenic action in previous assays with the Salmonella tester strains [9,10]. We have found that tests with both the Salmonella and the Bacillus cultures were negative within the practical solubility range of vinyl chloride in the nutrient broth under ambient conditions. Figure 1 shows the stability of a CMA 003754 ;h! oroox'i race . arc c~ :r:a: a as , wC.r is 3 ..ar a ^ r. ~j luOu-i 9joO fiame ion iZiv iCn 'jiS 'v.iru. av'w'j/'..^.1. r\ .. or, a `-cr. -a j-'U wit ",r. w i\ re ii 0-/ packed column was used at 30C for vinyl chloride determination. A 20% Curb;,',ax cn dromosorb ;c!u-'". at 53C and AC ml/mir, or" Helium was used for analysis of 4 and 5 unless specified otherwise. Both were 0.125 inch I\, "V x 5 feet stainless steel columns. Spectra v-'e-e obta-'red usi^t a Varian A-SO A and a Perkin timer R-12 spectrams-er. Solutions of 0z,0 and CMSO-do. were used with 3-(trimethylsilyl)propanesulfonic acid sodium salt as internal reference. Tetramethylsilane was used as a reference in COCl^ and CCl^. Probe temperature v/as 38 "C. RESULTS AND DISCUSSION Mutagenicity assays with Bacillus and Salmonella for compounds 1-9 are Tv Tv summarized in Table III. They are grouped into three categories in subsequent discussion. Further testing data are included in Tables IV-VI and Figures 2-4. Nonmutagenicity of vinyl chloride, chloroethanol, and chloroacetic acid Only high concentrations of vinyl chloride (20% v/v in air) have produced mutagenic action in previous assays with the Salmonella tester strains [9,10]. We have found that tests with both the Salmonella and the Bacillus cultures were negative within the practical solubility range of vinyl chloride in the nutrient broth under ambient conditions. Figure 1 shows the stability of a 003 755 presaturated vinyl chloride broth solution at 25C and 1 atm. The initial concentration of 0.022 M of vinyl chloride 1 rapidly decayed by 50% in 15 hr. Thereafter the escape of 1 from the broth slowed considerably. In the next 45 hr there was a further decline of only 18%. Thus the bacteria strains (B^. subtil is MC-1 and Salmonella TA 100) were exposed to the stabilized solution of 0.0106 M (723 ppm) of vinyl chloride in the nutrient broth. The negative activity observed was not unexpected since vinyl chloride lacks the electrophilic character common to many mutagens [23]. Although the direct mutagenicity of vinyl chloride at 200,000 ppm (20% v/v in air) observed previously [10] may be real, the chronic human exposure problem most likely requires metabolic activation of vinyl chloride to an electrophilic reactive form [8]. Also tested were chloroethanol 2 and chloroacetic acid 3 which probably are metabolic intermediates as shown in Scheme I. Both S-(2-hydroxyethyl)cysteine and SCHEME I: Vinyl Chloride Metabolites CH, CHC1 1 cellular C1CH2-CH20H 2 SH cellular C1-CH2-C02H 3 SH _o2c-ch(nh3+)-ch2 ho-ch2-ch2-s s(ch2-co2h)2 thiodiglycolic acid in Scheme I have been identified [6] as the urinary metabolites of vinyl chloride. Neither chloroethanol 2 nor chloroacetic acid 3 exhibited any mutagenic effects at 1 mM concentration in our mutagenicity assays (cf. Fig. 2 ). Although Bartsch et_al,[10] found'considerable mutagenic CMA 003756 11 activity with chloroethanol 2 for TA 1530 strain in the absence of microsomal 'V activation, our observations corroborate with those of McCann et al_. [7] who showed that 2 was weakly mutagenic directly even at high concentrations for /V the sensitive TA 100 and that it showed only trace activity for TA 1535. The enhanced activity of 2 after microsomal activation observed by these groups suggests that chloroacetaldehyde derivatives were being formed from chloro ethanol. We have found potent mutagenicity and lethality with the various forms of chloroacetaldehyde as shown in Figures 2-4 and Tables IV-V. Mutagenesis of chloroacetaldehyde, the monomer hydrate, dimer hydrate, and the trim- er - When chloroacetaldehyde *5\t was distilled into distilled v/ater, a mixture of the monomer hydrate 6 and the dimer hydrate 7 was formed instantly as determined by HPLC and PMR spectroscopy. Analysis of the commercial 45% aqueous chloroacetaldehyde solution with the same techniques showed a 50:50 mixture of the twohydrates. Upon standing under dry conditions, the monomer 'V5 cyclized to form trimer *8\#.The trimer was sparingly soluble in water, but was disproportionated upon heating in water to form the hydrates '6V and *7\t . Purified samples of chloroacetaldehyde 5, the commercial 45% chloroacetaldehyde solution containing a 50:50 mixture of the hydrates 6 and 7, the dimer hydrate 7, and the trimer 8, in DMSO solutions were tested for mutagenic '\i '`Vi potential. The data in Table IV summarizes the results of the repair assays with '-B-. "s--u btilis. Chloroacetaldehyde 5 and\ the monomer hydrate <\6i specifically inhibited the growth of B_. subtilis MC-1, a mutant lacking recombination repair of DNA. These unrepaired DNA lesions then led to cell death. However, compounds 5 and 6 did not inhibit the wild type EH subtilis or those mutant CMA 003757 aci'.V; :y wr:n cr.iorce;r.anui J - 'J 5 -F * - i . s'ct'.'/ai'.o.", c i r coservaoio-s ^ ,:rzz'- ;f showed that 2 was weah> r/,:-;'.-;;: directly even at high ct.-,-er:-ations the sensitive TA ICO and that it showed only trace activity for TA 1535. The enhanced activity of 2 after microsomal activation observed by these groups suggests that chloroacetaldehyde derivatives were being formed from chloro- ethanol. We have found potent mutagenicity and lethality with the various forms of chloroacetaldehyde as shown in Figures 2-4 and Tables IV-V. Mutagenesis of chlcroacstaldehvde, the monomer hydrate, dimer hydrate, -a-nd the trimer - When chloroacetaldehyde A5* was distilled into distilled water, a mixture of the monomer hydrate *6* and the dimer hydrate A7# was formed instantly as determined by HPLC and PM.T spectroscopy. Analysis of the commercial 45S aqueous chloroacetaldehyde solution with the same techniques showed a 50:50 mixture of the two hydrates. Upon standing under dry conditions, the monomer i\5, cyclized to form trimer A8 . The trimer was sparingly soluble in water, but was disproportionated upon heating in water to form the hydrates 6 and 7. A, A, Purified samples of chloroacetaldehyde 5, the commercial 4525 chloroAi acetaldehyde solution containing a 50:50 mixture of the hydrates 6 and 7, the A, A, dimer hydrate A7* , and the trimer A8# , in DMSO solutions were tested for mutagenic potential. The data in Table IV summarizes the results of the repair assays with B. subtil is. Chloroacetaldehyde 5 and the monomer hydrate 6 specifically A* A# inhibited the growth of B_. subtil is KC-1, a mutant lacking recombination repair of ONA. These unrepaired DNA lesions then led to cell death. However, compounds 5 and 6 did not inhibit the wild type B_. subtil is or those mutant A# A# CMA 003758 strains (Hcr-9, FB-13) having the capacity for recombination repair. In contrast, purified samples of the dimer hydrate 7 and trimer 8 inhibited all -V 'V of the mutants as well as the wild type, although MC-1 again demonstrated the most sensitivity. It is unlikely that 7 and 8 caused DNA lesions that are different from those by 5 and 6 because the excision repair mutants and the wild type were inhibited to an equivalent extent. Inhibition of these strains by 7 and 8 may result from a metabolic poisoning or cellular damage similar to that observed in Escherichia coli after exposure to vinyl chloride waste [24]. Viability assays on G. subtil is strains during short term exposure to the chloroacetaldehyde 5 revealed that recombination repair was essential for recovery (Figure 2). The E_. subtil is strains with recombination repair cap abilities displayed repair kinetics as evidenced by the shoulders in curves in Figure 3. The sensitive MC-1 strain lacking recombination repair yet having excision repair was rapidly killed following the exposure. We surmise that these DMA lesions caused by the chloroacetaldehydes were repaired solely by the recombination repair mechanism. The direct mutagenic potential of the samples4-9 was determined by the mutations expressed in Salmonella strain TA 100. The dose response relation ships of these compounds (cf. Table V), and the dose-response curves (cf. Figure 4 ) were determined with strain TA 100. At high concentrations the dose response curves for all compounds became nonlinear because the toxicity of the chemicals reduced the number of potential revertants on the plates,. The reduction of the cell population by high concentrations was confirmed by viable counts and observations of decreased background lawn on the test plates. CMA 003759 Data from the dose response curves (Figure 4 ) showed that chloroacetaldehyde 5 and the monomer hydrate 6 were more active than 7 and 8. ^ ^ T, -V The monomer %5 was the most mutagenic as predicted because its electrophilic carbonyl group remains intact. The restoration of the carbonyl character to the monomer hydrate 6 can be projected in hydrophobic environment of the cell membrane by loss of water, hence its mutagenicity is also explicable. The substantial activity of the dimer hydrate 7 can be attributed to its ready equilibrium with the monomer hydrate 6 in aqueous medium. The relationship of the activity of the cyclic trimer 8 to the chloroacetaldehyde action cannot be deduced on the basis of the curves in Figure 4. Nevertheless, these mutations were the base-substitution type and were expressed in Salmonella strains TA 100 and TA 1535. The mutagenic response in TA 1535 v/as very weak as compared to TA 100. It should be noted that the latter contains an "R" factor that enhances mutation by an error-prone recombination repair mechanism following DNA damage [7], Comparison of mutagenic response of chlorooxirane with its methylene homolog epichlorohydrin - The prevailing opinion [s] is that chlorooxirane 4 is the primary metabolite of vinyl chloride and is derived from the action of the microsomal mixed function oxidase. This compound, prepared independently from chlorination of ethylene oxide, was found to rearrange readily to chloro acetaldehyde in aqueous or DMSO solution at ambient temperatures. A kinetic study of a 0.15 M solution of 4 in a D20-DMS0-d, (80:20) mixture at pD 7.1 and 4C by PMR technique showed that the rearrangement followed first order kinetics, k (sec"1) = 2.5 x 10"4. This translatesto a half life of 46.2 min CMA 003760 r.ri~3 i v.are more active than 7 and 8. 'X, The monomer 5 was the most mutagenic as predicted because its electrophilic carbonyl group remains intact, The restoration of the carbonyl character to the monomer hydrate o can be projected in hydrophobic environment of the 'V cell membrane by loss of water, hence its mutagenicity is also explicable. The substantial activity of the dimer hydrate 7 can be attributed to its 'Xi ready equilibrium with the monomer hydrate 5 in aqueous medium. The relationship of the activity of the cyclic trimer 3 to the chloroacetaldehyde action cannot be deduced on the basis of the curves in Figure 4. Nevertheless, these mutations were the base-substitution type and v/ere expressed in Salmonella strains TA 100 and TA 1535. The mutagenic response in TA 1535 was very weak as compared to TA 100. It should be noted that the latter contains an "R" factor that enhances mutation by an error-prone recombination repair mechanism following DMA damage [7], Comparison of mutagenic response of chlorooxirane with its methylene homoloq epichlorohydrin - The prevailing opinion fsl is that chlorooxirane 'X4i is the primary metabolite of vinyl chloride and is derived from the action of the microsomal mixed function oxidase. This compound, prepared independently from chlorination of ethylene oxide, was found to rearrange readily to chloro acetaldehyde in aqueous or DMSO solution at ambient temperatures. A kinetic study of a 0.15 M solution of 4 in a D?0-DMS0-d, (80:20) mixture at pD 7.1 and 48C by PMR technique showed that the rearrangement followed first order kinetics, k (sec"^) = 2.5 x 10"4. This translates to a half life of 46.2 min 003T61 at 4C as compared to 1.6 min at 37C [9]. Such instability allows only limited testing in the cold as well as interpretation of the results. On the other hand, epichlorohydrin 9, which can be considered the epoxidation metabolite of allyl chloride, is a methylene homolog of 4, Both 4 and 9 are structurally 'V Oj 0* bis-alkylating agents, hence comparing their mutagenic activities will lend insight into the mode of action of chlorooxirane 25,26], Table IV tabulates the mutagenic response of these two compounds in the Bacillus repair assay and Table VI the Salmonella TA 100 reversion. Due to the instability of chlorooxirane at 37C, the assays shown in Table VI were preincubated at 3C in solution with the Salmonella TA 100 strain before plating to insure that chlorooxirane could reach the cells intact. Mutant strains of B_. subtil is were not inhibited when exposed to high concentrations of epichlorohydrin 9. In contrast, chlorooxirane 4 selectively inhibited the rec~ strain MC-1 in a manner similar to chloroacetaldehyde hydrate 6. Tests with Salmonella showed that strain TA 100 was very susceptible to the mutagenic action of both epoxides 4 and 9. However 9 was less toxic to the tester strains than 4. The low toxic effects of 9 indicate a different type of DyNA lesion compared to that caused by chlorooxirane 4. It is possible that 9 may react with DNA by a mechanism which does not cause potential lethal strand-scissions. On the other hand, chlorooxirane 4 may act on the bacteria via a NIH shift [27,28] to form chloroacetaldehyde 5 or 6. Conceivably, chlorooxirane can also behave as a diradical intermediate rather than a conventional $1 or SM2 type alkyl- N ating agent in its reaction with DNA. Vinyl chloride carcinogenesis mechanism hypothesis - Among the comoounds tested in the metabolic Scheme II, chloroacetaldehyde 5 and chlorooxirane 4 A# ' 'V CMA 03 762 .3 SCHEME II: The metabolic pathways of vinyl chloride [5], I. C1-CH=CH~ 1- Cl -CIL-CIL-OH 2- SCHEME I urine alcohol Cl-CH2-Ch'0 5 dehydrogenase C1-CH2-CH0 5- C1-CH2-C02H 3 'U SCHEME I urine II. H22 Cl-CH2,,-CHZ9-OH 2^ ------c--a- t7a7l ase ci-ch2-ch2-ooh Cl-CHo-CHO 5 ^ 'V, III. C1-CH=CHc9 %1 oxidase Cl-CH-CH-0 4 t2j * C1-CH--CK0 5 v/ere the most mutagenic with the lowest toxic side effects. Hence, they may qualify to be the active carcinogenic derivatives of vinyl chloride. Consider ing the aqueous milieu of the metabolic environment, however, the chloroacetal dehyde monomer hydrate 6 is a more realistic choice as an ultimate carcinogen than the monomer 5 which reacts immediately with water. Viability assays following exposure of the B^. subtil is mutants to 6 indicated that the compound induced recombination repair. The strain with the rec phenotype (MC-1) was immediately inactivated. All the rec+ strains showed survival ability and repair kinetics of similar nature. It has been shown [27] that mammalian cells have postreplication repair of DNA which has many similar features to the recombination repair in bacteria. In addition, a relationship between this mammalian postreplication repair process and mutation as well as carcinogenesis 003T63 ut SCHEME II: The metabolic path-,/;y: of vinyl chloride [5j. ui-^H=Un, C1-CH2-CH0 5'v ; <2"'2' C1-CH2-C02H 3 jr;re 31c^ho1 ----------------- -ci-ck2-cho 5 dehydrogenase ^ SCHEME I urine II. Ci-ch2-ch2-oh \ H22 Cdla dSC r' rj r>^u , ' r l? r i_n c: r\. III. C1-CH=CH^- "1u oxidase Cl -CH-C.H,,0 4 Cl-ch2-cho were the most mutagenic with the lowest toxic side effects. Hence, they may qualify to be the active carcinogenic derivatives of vinyl chloride. Consider ing the aqueous milieu of the metabolic environment, however, the chloroacetaldehyde monomer hydrate 6 is a more realistic choice as an ultimate carcinogen % than the monomer 5 which reacts immediately with water. Viability assays following exposure of the B. subtil is mutants to 6 indicated that the compound induced recombination repair. The strain with the rec" phenotype (flC-1) was immediately inactivated. All the rec+ strains showed survival ability and repair kinetics of similar nature. It has been shown [27]that mammalian cells have postreplication repair of DMA which has many similar features to the recombination repair in bacteria. In addition, a relationship between this mammalian postreplication repair process and mutation as well as carcinogenesis CMA 003764 has been suggested 4]. Cells from patients with the skin disease xeroderma pigmentosum lack the ability to excise pyrimidine dimers and must rely on postreplication repair to remove these lesions [24]. It is believed that this error prone process of postreplication repair is responsible for the production of somatic mutations and cancer in patients with this disease. Since chloroacetaldehyde monomer hydrate 6 induces recombination repair in bacteria responding to lesions, it may also be capable of activating the error prone postreplication repair in exposed mammalian cells. At the molecular level, chloroacetaldehyde is known to react with N1 and N6 nitrogensof adenosine and N3 and N4 nitrogensof cytidine in single-stranded DNA [28,29], It therefore appears that chloroacetaldehyde monomer hydrate 6 should merit our consideration as an ultimate carcinogenic metabolite of vinyl chloride. The lower mutagenic activity of chloroxirane 4 compared to 6 may reflect \y 0, the unstable nature of chiorooxirane as an a-chloroether. While the carcino genic chloromethyl methyl ether is a bifunctional alkylating agent [30], the mutagenic activity of chlorooxirane cannot be so categorized, especially when it is compared with epichlorohydrin 9. One mode of action of chlorooxirane is a rearrangement to chloroacetaldehyde via .the MIH shift [27,28]. Another is a homolytic ring cleavage to yield a stabilized diradical inter mediate CICH-CHgO. Both are capable of reacting with DNA, thereby accounting for the mutagenicity of 4. In mammalian cells, chlorooxirane,being a reactive 'V epoxide, could be trapped by a glutathione epoxide transferase [31] at a faster rate than the detoxification of chloroacetaldehyde via the less active aldehyde dehydrogenase [32] . Such detoxification of chlorooxirane could CMA 003765 explain the reported decrease in sulfhydryl level in liver cells during metabolism of short chain halo hydrocarbons including vinyl chloride [31]. Perhaps the lower mutagenicity of chlorooxirane 4 in these bacterial assays, compared to the chloroacetaldehydes, is also attributable to its being detoxified faster. We therefore consider both chlorooxirane 4 and the chloroacetaldehyde monomer hydrate 6 to be the ultimate carcinogenic metabolites of vinyl chloride. 'V Their reactions with DNA causing mutations in Bacillus and Salmonella suggest a causal relationship with vinyl chloride carcinogenesis in human and laboratory animals. Acknowledgements This work was supported by grants from the B. F. Goodrich Co. This grant program was initiated and administered by the Cancer Center of the University of Louisville. The Salmonella tester strains were kindly provided by Dr. B. N. Ames of the University of California, Berkeley. We also thank George D. Stratton, Or. and S. E. Yen for their able assistance. CMA 003766 explain the reported decrease in sol fry try! level in liver cells curing metabolism of short chain halo hyc.-ocarocns including vinyl cr.l price {3;1. Perhscs the lower mutagenicity of chlorooxirane 'V4* in these bacterial assays, compared to the chloroacetaldehydes, is also attributable to its being detoxified faster. We therefore consider both chiorooxirane 'V4 and the chlcroacetaldehyde monomer hydrate 6 to be the ultimate carcinogenic metabolites of vinyl chloride. Their reactions with ONA causing mutations in Bacillus and Salmonella suggest a causal relationship with vinyl chloride carcinogenesis in human and laboratory animals. Acknow!eagements This work was supported by grants from the B. F. Goodrich Co. This grant program was initiated and administered by the Cancer Center of the University of Louisville. The Salmonella tester strains were kindly provided by Or. 3. N. Ames of the University of California, Berkeley. We also thank George D. Stratton, Jr. and S. E. Yen for their able assistance. CMA 003767 REFERENCES AND FOOTNOTES I VI *To whom correspondence should be addressed. 1 Viola, F. L., Bigotti, A. and Caputo A. (1971) Cancer Research 31, 516-523 2 Haltoni, C. and Lefemine, G. (1974) Environmental Research 7, 387-405 3 Creech, J. L. and Johnson, M. N (1974) Journal of Occupational Medicine 16, 150-151 4 Lee, F. I. and Harry, D. S. (1974) Lancet 1, 1316-1318 5 Hefner Jr., R. E., Watanabe, P. G. and Gehring, P. J. (1975) Annals of New York Academy of Science 246, 135-148 6 Watanabe, P. G., McGowan, G. R. and Gehring, P. J., Annals of New York Academy of Science, in the press 7 McCann, J., Sinrnon, V., Streitwieser, D. and Ames, B. N. (1975) Proceedings of National Academy of Science U.S.A. 72, 3190-3193 8 Rannug, U.,Johnsson, A., Ramel, C. and Wachtmeister, C. A. (1974) Ambio 3, 194-197 9 Malaveille, C., Bartsch, H., Barbin, A., Camus, A. M. and Montesano, R. (1975) Biochemistry Biophysics Research Communication 63, 363-370 10 Bartsch, H., Malaveille, C. and Montesano, R. (1975) Internationl Journal of Cancer 15, 429-437 11 Hayduk, W. and Landle, H. (1974) Journal of Chemical Engineering Data 19, 253-257 12 Walker, G. (1953) Formaldehyde, p. 86, Reinhold, New York 13 Natterer, K. (1882) Monatsheffe fur Chemie 3, 443-464 CMA 003768 19 14 Harris, G., ed (1965) Dictionary of Organic Chemistry, Vol. 2, pp. 589 anc^F 956 15 McCann, J., Spingarn, N. E., Kobari, J. and Ames, B. N. (1975) Proceedings of National Academy of Science U.S.A. 72, 979-983 16 Okudo, S. and Romig, W. R. (1965) Journal of Molecular Biology 14, 130-142 17 Laumbach, A. D. and Felkner, I. C. (1972) Mutation Research 15, 233-245 18 Vogel, H. 0. and Bonner, D. M. (1956) Journal of Biological Chemistry 218, 97-106 19 Kada, T. Tutikawa, K. and Sadaie, Y. (1972) Mutation Research 16, 165-174 20 Walling, C. and Fredericks, P. S. (1962) Journal of American Chemical Society 84, 3326-3331 21 Gross, V. H. and Freiberg, J. (1969) Journal fur Praktische Chemie 311, 506-510 22 Zief, M. and Schramm, C. H. (1964) Chemistry and Industry April 18, pp. 660-661 23 Kappus, H. Bolt, H. M., Buchter, A. and Bolt, W. (1975) Nature 257, 134- 135 24 Hagstrom, A. (1974) Ambio 3, 77-79 25 Voogd, C. E. (1973) Mutation Research 21, 52-53 26 Fjellstedt, T. A. Allen, R. H., Duncan, B. K. and Jakoby, W. B. (1973) Journal of Biological Chemistry 248, 3702-3707 27 Lehmann, A. (1974) Life Science 15, 2005-2016 28 Barbin, A., Bresil, H., Croisy, A., Jacquignon, P. Malaveille, C., Montesar.o, R. and Bartsch, H. (1975) Biochemical and Biophysical Research Communications 67, 596-603 03769 14 Harris, G., 9 155} H->H sr.ar of 0 manic Cf.emi scry, 7c -7 * :?. =c; . . 15 McCann, J., Spmgarn, c., .\ooari, J. and k,.as, 3. H. rroceec,n55 of (National Academy of Science U.S.A. 72, 979-983 16 Okudo, S. and Romig, W. R. (1965) Journal of Molecular Biology 14, 120-142 17 Laumbach, A. D. and Felkner, I. C. (1972) Mutation Research 15, 233-245 18 Vogel, H. J. and 8onner, D. M. (1956) Journal of Biological Chemistry 213, 97-106 19 Kada, T. Tutikawa, K. and Sadaie, Y. (1372) Mutation Research 16, 155-174 20 Walling, C. and Fredericks, P. S. (1962) Journal of American Chemical Society 84, 3326-3331 21 Gross, V. H. and Freiberg, J. (1959) Journal fur Praktische Chem'e 311, 506-510 22 Zief, M. and Schramm, C. H. (1964) Chemistry and Industry April 18, pp. 660-661 23 Kappus, H. Bolt, H. M., Buchter, A. and Bolt, W. (1975) Nature 257, 134- 135 24 Hagstrom, A, (1974) Ambio 3, 77-79 25 Voogd, C. E. (1973) Mutation Research 21, 52-53 26 Fjellstedt, T. A. Allen, R. H., Duncan, B. K. and Jakoby, W, B. (1973) Journal of Biological Chemistry 248, 3702-3707 27 Lehmann, A. (1974) Life Science 15, 2005-2016 28 Barbin, A., Bresil, H., Croisy, A., Jacquignon, P. Malaveille, C., Montesano, R. and Bartsch, H. (1975) Biochemical and Biophysical Research Communications 67, 596-603 CMA 003770 20 29 Kochetkov, N. K. Shibaev, V. N., and Kost, A. A. (1971) Tetrahedron Letters 22, 1993-1996 30 Leong, B. K., Macfarland, H. N. and Reese, W. H. (1971) Archives of Environmental Health 22, 663-666 31 Johnson, M. K. (1965) Biochemical Pharmacology 14 , 1383-1385 32 Weiner, H., King, P., Hu, J. H. J. and Bensch, W. R. (1974) Alcohol and Aldehyde Metabolizing Systems (Thurman, R. G,, ed .), pp. 101-113, Academic Press, New York Ctfh LEGENDS TO FIGURES Fig. I. Stability of vinyl chloride in nutrient broth at 25C, 1 atm. Fig. 2. Survival of Bacillus subtil is MC-1 after incubation with: 1.0 nil chioroethanol 2 ( *J*--F); 1.0 mM cHToroacetic acid ^ (--); 5.76 n:M (concentra tion based on CIH2CCHO) chloroacetaldehyde (45% aqueous solution) 6 and 7 ( 0--0 0.1 mM 4-nitroquinol ine-N-oxide (0-0); and untreated control cel^Is (0 -- 0). Mid-logarithmic cultures grown in MY-1 broth were incubated with compounds at 37aC. Samples were plated at the times indicated from which viable cell counts were recorded. The mutagen 4-nitroquinoline-N-oxide served as a positive mutagenicity control. Fig. 3. Survival of Bacillus subtil is strains in the presence of 5.0 mM (concentration based on CIH2CCHO) chloroacetaldehyde (45% aqueous solution) j6 and 7. Cultures were grown to mid-logarithmic phase in MY-1 broth and then incubated with the chloroacetaldehyde solution at 37C. Samples were plated at the times indicated from which the percent survival was determined. FB-13 uvr~, rec+ (-h -- -F);Hcr-9 hcr", rec+ (0-0); 168M wild type (0 -- 0); and MC-1 uvr+, rec" (c>--o). Fig. 4. .Dose response curves with Salmonella typhimurium TA1QQ. Sample solution of known concentrations prepared in DMSO were mixed with tester strain culture and soft agar. Plates were poured, incubated at 37C for 48 hrs, and then sjare for revertant colonies to prototrbphy. Chloroacetaldehyde monomer 5 (o-c^p chloroacetaldehyde (45% aqueous solution-concentration based on CIH2CCKO) 6 and 7 (0-0); chloroacetaldehyde dimer hydrate .( i>--C>); chloroacetaldehyde trimer $ ( k--k); and epichlorohydrin (0--j). CMA. 003 772 lscemss to fig-.tzs rig. 1. 5tabi;ity c; -0 - Fig. 2. Survival of Caci11 us subtil is MC-1 after incubation v/ith: 1.0 r", chiorocthanol 2 ( *5*-^; 1.0 mM cnloroacetic acid 3 (--); 5.76 mM (concentra tion based on Cll^CCHO) chloroacetaldehyde (45% aqueous solution) 6 and ,7 ( t>--0 0.1 mM 4-ni troquinol ine-h'-oxide (0-0); and untreated control cells (0 -- 0). Mid-logarithmic cultures grown in MY-1 broth were incubated with compounds at 37*C. Samples were plated at the times indicated from which viable call counts were recorded. The mutagen 4-nitroquinoline-N-oxide served as a positive mutagenicity control. 3. Survival Gi S2C ( -L,w >. 1 i . o strains in th.e presence of 5.0 (concentration based on CihgCCriO) cnlorcacetaldehyde (45% aqueous solution) ,6 and 7. Cultures ware grown to mid-logarithmic phase in MY-1 broth and than incubated with the chlorcacetaldehyde solution at 37C. Samples were plated at the times indicated from which the percent survival was determined. F3-13 uvr", rec+ (:-------i-);Hcr-9 her", rec* (0--0); 7 63M wild type (0 -- 0); and MC-1 uvr+, rec" (> -- t>) - Fig. 4. .Dose response curves with Salmonella typhimurium TA100. Sample solution of known concentrations prepared in LiMSO were mixed with tester strain culture and soft agar. Plates were poured, incubated at 37C for 48 hrs, and then score for revertant colonies to prototrOphy. Chloroacetaldehyde monomer 5 (0--0); chloroacetaldehyde (45% aqueous solution-concentration based on ClHjCCHO) 5 and 7 (0-0); chloroacetaldehyde dimer hydrate ,.( J -- o); chloroacetaldehyde Primer jg (--); and epichlorohydrin 003773 Ci o Figure 1 C 0 aC EfiT gA 11 0 X M IL L ) m o la r 5_ l|L 0 12 JL 24 30 4S , CO TIME HOURS CMA 003774 Figiue 2 CMA 003775 I 0 iV GO MI till U S 90 120 r *=-` - - 003778 REVERT ANT COIOKIES PER PLATE / TABLE I: Bacteria Tester Strains A. Salmonella typhimurium IT-2 Tester Strains3 a All tester strains contain uvrB repair mutations which eliminate the excision repair system; mutations in the histidine operon; and rfa mutations which alter the cell wall by increasing permeability and eliminating pathogenicity. bThe resistance transfer factor,"R" factor,enhances the error-prone recombination repair system thus making the strains more susceptible to mutation [15]. cThe strains susceptible to base-pair substitution contain mutations in the histidine G46 operon and those susceptible to frameshift mutation contain mutations in the histidine operon C 3076 (TA 1537) or D3052 (TA 1538, TA 98). Strains "R" factorb Mutation detected0 TA 1535 TA 100 TA 1537 TA 1538 TA 98 base-pair substitution + base-pair substitution - frameshift - frameshift + frameshift B. Bacillus subtil is Tester Strains aTrp~ denotes a requirement for tryptophan; Mit-S denotes sensitivity to mitomycin C. bhcr+ denotes a host-cell reactivation DNA repair capacity; hcr~ lacks a host-cell reactivation DNA repair capacity; rec+ denotes a recombin ation DNA repair capacity; rec" lacks a recombination DNA repair capacity; uvr~ is sensitive to ultraviolet-induced DNA damage. Strains 168 M llcr-9 FB-13 MC-1 Phenotype3 Prototroph (wild type) Trp" Trp" Trp",Mit-S DNA Repair^ .her+ , rec+ her", rec+ uvr", rec+ her , rec CMA 003780 TABLE II. PMR Spectra of Vinyl Chloride Derivatives Compounds C1HC1_-C_HI-s0 *4 ClHgC-CHO 5 % C1H?C-CH(0H)o 6 oo oo -t* Sol vent DMSO-dg CD90D-DoQ 1:8 3(pD 6.1) j 1 Spectra, 6TMS=Q (0=K"1 2.75 (q, CH, 0=1.5) 2.85 (q, CH, J=2.4) 4.90 (q, CH, 0=2.4,1.5) 4.00 (d, CH, J=2.2) 9.57 (t, CH?, 0=2.2) 3.50 (d, CH) 9.60 (t, CH2) 3.60 (d, CHo, 0=5) 4.60 (t, CH, 0=5) C1H,C-CH-0H 11 0 C1H2C-C* H-0H 7' dmso-d6 CD-0D-Do0 1:8 J(pD 20.1) 3.55 (d, CH?, 0=4.9) 5.05 (t, CH, 0=4.9) 3.60 (d, CH9, 0=4.5) 4.83 (t, CH? 0=4.5) ch2ci o'^o i JL 1 r^0'^XH9Cl 22 8 ~ oO 1 DMSO-dg 3.52 (d, CH2, 0=4.7) 5.08 (t, CH, 0=4.7) 3.75 (d, CHo, 0=4.1) 5.45 (t, CH, 0=4.1) CMa 003781 TABLE II. ?XR Spectra -s1 ll.tfi fLk 1 t * `J w< .---J, Seri vet:ves CcmcoLir.ds C1HCl -CH2-0t ^4 ClHgC-CHO 5 C1H2C-CH(0H)2 6 So i vent cci4 CC14 DMSO-dg CD.OQ-DpQ 1:8 3{pD 6.1) Zzectro f j t . -- -- \j \ _ -- 1 . 2.75 (q, CH, 0=1.5) 2.35 (q, CH, 0=2,4) 4.90 (q, CH, 0=2.4,1.5) 4.00 (d, CH, 0=2.2) 9.57 (t, CH?, 0=2.2) 3.50 (d, CH) 9.60 (t. CH2) 3.60 (d, CK2, 0=5) 4.60 (t. CH, 0-5) C1H-C-CH-0H 1i 0 1 C1H2C-CH-0H 7 DMSO-Dg CD.OD-D 0 1:8 3(pD 20.i) 3.55 (d, CH-, 0=4.9) 5.05 (t. CHf 0=4.9) 3.60 (d, CH9, 0=4.5) 4.83 (t, CHf 0=4.5) CHpCl A 1 (r^O^XH9Cl 8 2^ CC14 DMSO-dg 3.52 (d, CH2, 0=4.7) 5.08 (t, CH; J-4.7) 3.75 (d, CH9, 0=4.1) 5.45 (t. CHf 0=4.1) CMA 003782 TABLE III: Summary of Mutagen Activity in Microbial Systems Ni = No inhibition of growth detected in Bacillus subtil is MC-1; NR = No increase of revertants in Salmonella typhimurium iA 100 compared to control; + =active; ++ = very active. Acetaldehyde, a potential metabolite of and ally! chloride, the parent olefin of were negative in these two systems. Compounds Tested Control: 4-nitroquinoline-N-oxide -1\r Hc9C=CHC1 'X2, CIH.C-CH.OH L L- "X3. CULC-COOH L 4 C1HC-CH--0 'v 1 2 i 5 CULC-CHG 6 C1H,,C-CH(0H)? ^7 ClH,C-CH0H-0-CH0H-CH,Cl C. L. 8 (ClH2C-CK0-)3 9 ClHoC-CH-CH,0 ~ 2 i____ h Bacillus subtil is Repair Assay ++ NI NI NI + ++ ++ ++ ++ NI Salmonella typhimurium Reversion Assay ++ NR NR NR ++ ++ ++ ++ + ++ CMA. 003783 TABLE IV: Growth Inhibition of Bacillus subtil is Strains . Inhibition was measured in mm after 24 hr at 37C as described in text; MI denotes no inhibition Mutaqen C1H.C-CH0 5 Molarity 0.100 Chloroacetaldehyde & (45% aqueous solution) 0.115 CHLC-CH-QH 1 01 7i C1H2C-CH-0H 0.097 168M 2.0 NI 1.5 MC-1 27.7 22.5 9.5 Hcr-9 3.7 FB-13 2.7 NI NI 1.5 1.5 cih2c^\^ch2ci KJ 1 C1)2CI 8 ^ ClHC-CHpO 1___ Li 4 ~ CHL2C-CiH-CJ-L2Qi n9. 0.096 0.260 0.113 4-Nitroquinoline-N-oxide 0.001 (control) 7.0 14.5 6.0 NI NI 10.0 10.0 NI 18.0 NI NI 15.0 NI NI 15.0 CMA 003784 innibition was nieas'jred in "'H after 24 nr at 37C M denotes no inhibition GtfSCi' '** tfutaaen Molarity C1HC--C-CH0 ^5 0.100 Chlcroacetdldehyde 5 i ,7 (*r z 'm 5 ^ - 3 c l.' j s c j u u i o n} 'v * 3 ClJLC-CH-CH ` 0' 1 7n, C"> C-CH-0H 0.097 1 QCt'i MC-l Her-9 F5-1 i 2.0 27.7 3.7 2.7 Hi 22.5 Ui 11 * 1.5 9.5 1.5 1.5 ci}:2c^\^cn2ci V 1 C:12 C l 8 ^ C1HC-CH70 1___ =J 4 * C1H2,C-Ci H-CH2,01 %9 0.096 0.260 0.113 4-Nitroquinoline-N-oxide 0.001 (control) 7.0 NI MI 10.0 14.5 6.0 7.0 10.0 NI NI NI NI NI 18.0 15.0 15.0 CMA 003785 30 TABLE V. Relative Mutagenicity of the Four Forms of Chloroacetaldehyde with s.typin'murium TA 100 45% Aqueous Soln 6 : 7 50:50 Monomer 5 ~ Dimer Hydrate 7 ~ Trimer 8 ^ Molarity 5.3xl0"5 2.7x10" 1.4xl0"5 6.9x10" 3.4x10~ 1.7x10" 8.6x10"7 4.3x10"7 Revertants Molarity 977 1.3xl0-5 723 6.7xl0'6 512 3.3xl0"6 194 1.7x10" 120 8.4x10"7 61 4.2xl0"7 36 2.1xl0'7 10 l.lxlO-7 Revertants 18 68 88 361 404 238 185 131 Molarity 4.8x10"4 2.4xl0~4 1.2xl0-4 6.0x10" 3.0x10" 1.5x1 O'5 7.5x1 O'6 3.8x1 O'6 Revertants 311 259 193 107 88 30 23 11 Molarity 4.8xl0"4 2.4xl0"4 1.2xl0"4 6.0x10" 3.0xl0"5 1.5x10"5 7.4x10" 3.7x10" Revertants 144 159 101 39 27 18 12 -0 CMA 003786 J I TABLE VI: Reversion of S.typhi murium TA100 by Chiorooxirane 4 and Epichlorohydrin 9 % Broth solutions of ^ or ^ with TA100 v/ere preincubated at 3C before plating. Duplicate plates were evaluated after 48 hrs at 37C.' The average number of revertant colonies per plate minus the number of spontaneous reversions were recorded. Preincubation Time 0 hr 1 hr 2 hr 4 hr 6 hr Epichlorohydrin 9 (1.0 mM) ^ 186 204 297 202 154 Chiorooxirane 4 (0.26 mM) % 31 4 6 114 44 CMA 003787 "1 , , , _ ^ J Broth solutions of ^ or $ 'with 7A100 were preincubated at 3C berore plating. uupticar, e p t a l e s i*c e v a 1 u a l od a* ter 43 nrs at 37C." The average number of revertant colonies per plate minus the number of spontaneous reversions were recorded. Preincubation Time 0 hr 1 hr 2 hr ' 4 hr 6 hr Epicnlorohydrin 5 !i _o 1 % 13S 204 297 202 154 Chlorooxirane 4 CO.25 31 4 6 114 44 CMA 003788