Document DGpbGVzjMn72yyMEOENLa9mRB

E. P. Wheeler Lack of Cytogenetic Effects in IJone Marrow and Spcrmaingonial Cells in Hals Treated with Polychlorinated Biphenyls (Aroclors 124-2 and 1254)* by Sidney Green, Jacqueline V. Carr, Kenneth A. Palmer, and Elizadetii J. Oswald Division of Toxicology Food and Drug Administration Department of Health, ^duration, and Welfare . Washington, D.C. 20204 FEB 2 4 1975 The polychlorinated biphenyls (PCBs) are used primarily as coolants and insulators in the electrical industry (ANONYMOUS 1972), The stability of these compounds made them useful in the past as industrial stabilizers for paints, rubber, asphalt, printers' ink and pesticides. However, in recent years, the use of PCBs has been restricted to enclosed application, for example, in electrical capacitors and transformers. PCB's have gained worldwide recognition as pollutants of ecosystems. Traces of these compunds have been found in a\range of species from fish to man (HANSEN et al. 1971; JCOLBYE 1972; ZITKO 1972). There is increasing concern over the threat these environmental contaminants pose to human health; thi6 concern has its orlgla in the adverse reactions . observed after treatment of experimental animals with PCBs and after accidental exposure of humans to high levels of PCBs. Humans are exposed to the PCB's through contaminated air, water, and food (STAN0V1CK et al. 1973; IARRANT and TATTON 1968; VEITH and LEE 1970). In the "Yusho" incident in Japan, approximately 1,000 humane consumed rice oil contaminated with PCBs (KURATSUNE et al. 1972). PCBs have been found in human adipose tissue and in human milk (BIROS et al. 1970). The PCBs exert a variety of biological effects. The most common subjective symptoms described by male and female "Yusho" patients were dark brown pigmentation of nails, acne-like skin eruptions, pigmentation of the skin, increased eye discharge, hyperemia of the conjunctiva, and swelling of the upper eyelids (KURATSUNE et al. 1969). Studies in rats have shown that the PCBs induce several liver microsomal enzymes (ALVARES et al. 1973; BRUCKNER et al. 1973; LITTERST et al. 1972), reduce the urinary levels of estrogen and dehydroeplandrosterone in the boar (PLATONOW et al. 1972), produce hyperplasia and dysplasia of the gastric mucosa in male Rhesus monkeys (ALLEN and NORBACK 1973), increase host susceptibility to viral infection (FRIEND and TRAINER 1970), *Ihis manuscript was presented in part at the Twelfth Annual Meeting of the Society of Toxicology, New York, N.Y., March 18-22, 1973. 14 fcttllrt]* *1 E*lrefnra(j| Ccaismtaatlon 4 Toiicolofy, Yki. 1), N*. I 1975 by 5pft*|ri-Vc(U| New Yoik lac. DSN 026337 STLCOPCB4010299 1 m rily t then i In :loted In ver a Ions Bs and Is. , water, ; veith Imstely adipose most ited % of Its :rst i. n ),>st 0 T ^.Wid l synergize the :oxii ity of pesticide residues (LICHTENSTEIN et al. 1969), and inhabit the growth of human cells In culture (LITTERS! and TENSTEIN 1971). In studies of the reproductive effects of PCBs, KEPL1NGER et al. (1971) fed chickens 10 or 100 ppra of Aroclor 1242 or 100 ppm of Aroclor 1254 and reported loss of body weight, decreased thickness of egg shells, and poor hatchabllity of eggs. KIMBROUGH (1971) reported that fewer offspring were bom after Aroclor 1254 was fed to rats at 100 ppm, and that the pups born vere smaller and had decreased survival time as compared with the control group. Fetal loss, as exemplified by fewer offspring, is consistent with potential dominant lethal effects. The latter have as a basis cytogenetic damage (EPSTEIN et al. 1972). This report presents the results of the cytogenetic investigation of bone marrow and spermatogonial cells of rats after their treatment with Aroclors 1242 and 1254. Materials and Methods Aroclor 1242 was obtained from Mr. W.B, Fopageorge of Monsanto Industrial Chemicals Co. and Aroclor 1254 was obtained from Mr. Sidney Williams of the Chemical Technology Branch of the Food and Drug Administration. Both samples, according to information supplied by Monsanto, were free of contamination by the dibenzofurans. i Random-bred male Osborne-Mendel rats, weighing 180-250 g were randomly assigned to groups of eight. Aroclor 1242 was given orally at single doses of 1250, 2500, or 5000 mg/kg, or as a multiple dose of 500 mg/kg/day for 4 days. Our usual 5day regimen was not accomplished because of the debilitated condition of the animals after the compound had been administered for 4 days. Aroclor 1242 was administered as an undiluted solution at 5000 mg/kg and as a solution in com 0,11 at the other dosages. Aroclor 1254 in corn oil was given in a multiple regimen for 5 days at doses of 75, 150, or 300 mg/kg/day. Controls received corn oil. The rats were killed 24 hours after the single doses or 24 hours after the last dose in the multiple regimen. Three hours before killing, Colecimid was injected Intraperltoneslly at a dose of 4 mg/kg. Bone marrow was aspirated from both femurs and processed according to the method of LEGATOR et al. (1969) with a slight modification of the incubation time; in this investigation the cells were allowed to incubate in the hypotonic solution for only 20 minutes. Spermatogonial cells were processed according the method of H00 and BOWLES (1971). One hundred cells per animal (50 from each of two slides) were examined IS wiy wrsaaawn, wj 'wmwgew im mi i DSM 026338 i STLCOPCB4010300 for chromosomal abnormalities and one thousand cells per animal (500 per slide) for mitotic inhibition. Results from the treated groups wore compared with control values by a _t-test. For bone marrow and spermatogonial aberrations, the individual proportion of affected cells for each animal was transformed to the Freeman-Tukey arc sine to satisfy the assumptions of the .t-test (MOSTELLER and YOUTZ 1961). The transformations were not done for mitotic inhibition because the sample size was adequate to satisfy _t-tcst assumptions. The spermatogonial procedure was not performed with rats given Aroclor 1254, since Aroclor 1242, the more tonic of the compounds, did not produce chromosomal aberrations in spermatogonia. In addition, it was felt that a companion dominant lethal investigation of both compounds would yield more definitive information about germ cell effects. Results Toxicity Table 1 shows that the administration of Aroclor 1242 at 1250 mg/kg did not cause the death of any animals but body weight was reduced an average of 5 g. In the group given 2500 mg/kg, 4 of 8 rats expired within 24 hours and in the group given 5000 mg/kg, 3 of 8 expired. Weight losses in these groups were 20 and 26 g, respectively. The multiple doses of 500 mg/kg/day for 4 daj/s caused the death of 5 of 8 rats within 96 hours after the first administration and a mean weight los6 of 11 g, showing the cumulative toxic effect of this compound. table i Toxicity of Aroclor 1242 and 1254 after Oral Administration to the Osborne-Mendel Rat Group Control Aroclor 1242 Aroclor 1242 Aroclor 1242 Aroclor 1242 Control Aroclor 1254 Aroclor 1254 Aroclor 1254 Dose (mg/kg) Corn oil 500 X 4 1250 X 1 2500 X 1 5000 X 1 Corn oil 75 X 5 150 X 5 300 X 5 No. Dead/ No. Treated 0/8 5/8 0/8 4/8 3/8 0/8 0/8 0/8 1/8 Mean Weight . Loss (g) 11 5 20 26 . - 18 34 16 WI'WIU'EHIT*' DSW 026339 4J. 4* from the VI* rats f eld 2 d in , tlplc of 8 i mean of tratlon Weight 11 5 20 26 18 34 Aroclor 1254, although producing very few deaths, did cause a reduction in weight which exceeded that of the rata treated with Aroclor 1242, The subacute doses of 150 and 300 mg/kg produced a mean reduction in weight of 18 and 34 g, respectively (Table 1). Mitotic Inhibition The administration of Aroclor 1242 had no demonstrable effect on mitotic division of rat bone marrow cells (Table 2). TABLE 2 Number of Bone Marrow Cells with Abnormalities and in Mitosis after Treatment of Individual Male Osborne--Mendel Rats with Aroclor 1242 Aroclor 1242 (mg/kg) 0 Total proportion 500 X 4 Total proportion 1250 X 1 Total proportion 2500 X 1 Total proportion 5000 X 1 Total proportion OO oo oo o Number of Cells with Chromosomal Abnormalities^ 1. 1, 0, 1. 0, 0, 1, 0 4/800-0.005 0, 0, 0, 0, 1, 1, 0, 0 2/800-0.003 0, 1, 5, 0, 0 6/500=0.012 Number of Cells in Mitosis^ 42, 38, 32, 22, 44, 64, 40, 40 322/8000-0.040 38, 32, ?7 97/3000=0.032 44, 57, 62, 54, 32, 28, 24, 24 325/8000=0.041 54, 60, 26, 26 166/4 000=0.042 28, 45, 38, 50, 45 206/5000=0.041 =One hundred cells examined per animal. tone thousand cells counted per animal. Although the number of mitotic cells in rats given the multiple doses of 500 mg/kg was reduced, the decrease was not atetistically significant. As seen in Table 3, Aroclor 1254 at 75 mg/kg did not inhibit mitosis in bone marrow but doses of 150 and 300 mg/kg produced indexes of 0.029 and 0.031, respectively, as compared with a control index of 0.041. 17 l DSW 026340 1 OBW ' STLCOPCB4010302 TABLE 3 Number of Bone Morrow Cells with Abnormalities and in Mitosis after Treatment of Individual Hale Osborne-Mendcl Rats with Aroclor 1254 Aroclor 1254 issAa)..... .......................... 0 Total proportion 75 X 5 Total proportion 150 X 5 Total proportion 300 X 5 Total proportion Number of Cells with Chromosomal Abriormalit les-- 1, 0, 0, 1, o. 1, 0, 1 4/800=0.005 1, 0, 1, 0, 0, 1, 1, 0 4/800=0.005 0, 0, 0, 1, 0, 1, 0, 0 2/800=0.003 0, 1, 1, 1, 0, 1, 0 4/700=0.006 Number of Cells in Mitosis-- 35, 40, 38, 45, 60, 38, 40, 35 331/8000=0.041 61, 46, 40, 24, 38, 42, 36, 29 316/8000=0.040 12, 38, 22, 40, 40, 30, 18, 35 235/8000=0.029^ 48, 28, 40, 16, 32, 26, 29 219/7000*0.031-- ^One hundred cells examined per animal, ione thousand cells counted per animal. ^Significantly different from control value at P<0.05, onetail J:-test. Table 4 shows that at acute dosages of 1250 and 2500 ng/k*. Aroclor 1242 had no effect on mitosis of sperraatogonial cells but 5000 and 500 mg/kg x 4 significantly decreased the rate of division. Chromosomal Abnormalities There vae no evidence that Aroclor 1242 administered to rata at 1250 or 2500 mg/kg produced a significant number of chromosomal aberrations in bone marrow cells (Table 2). In the group given 5000 mg/kg, the number of abnormalities appeared to be increased, but the result was considered Insignificant inasmuch as the response was due mainly to one animal. The subacute dosage of 500 mg/kg also failed to produce chromosomal damage. All lesions seen were of the single chromatid type. 18 DSW 026341 STLCOPCB4010303 Kit os Is ' with >f Cells >ls 38, 45, AO, 35 *"0.041 40, 24, 36, 29 *0,040 22, 40, 18, 35 `0.029 AO, 16, 29 *'0.031- one- 00 togonial ed the ed to er of . In i 1 to one to the i TABLE 4 Number of Spcrmatogonial Colls with Abnormalities and In Mitosis after Treatment of Individual Male Osborne-Mendel Rats with Aroclor 1242 i Aroclor 1242 (mg/kp,) ____________ Number of Cells with Chromosomal Abnormalities^- Number of Cells In Mitosis^. 0 Total proportion 0, 0, 0, 2/68, 1. 0, 0, 0 3/768-0.004 11, 18, 14, 2, 13, 15, 28, 18 119/8000=0.015 S00 X 4 Total proportion 0, 2, 1 3/300=0.010 A, 8, 8 20/3000=0.007 I 1250 X 1 0, 0, 1, 1, 0, 0, 0, 0 8, 5, 12, 22, 18, 20, 6, 11 Total proportion 2/800=0.003 102/8000=0.013 I 2500 X 1 0, 1, 1, 0 20, 20, 12, 18 i Total proportion 2/400=0.005 70/4000=0.018 5000 X 1 Total proportion 0, 1, 3, 0, 0 4/5dO=0.008 10, 12, 6, 8, 9 45/5000=0*. 009^ One hundred cells examined per animal, tone thousand cells counted per animal. ^Significantly different from control value at jKO.Ol, two tailed _t-tcst. ^Significantly different from control value at P<0.05, two tailed _t-test. Aroclor 1254 failed to produce a significant number of chromosomal abnormalities in bone marrow at the dosages tested. The proportions of abnormal cells were 0.005, 0.003, and 0.006 for the groups given 75, 150, and 300 mg/kg, respec tively (Table 3). No cytogenetic abnormalities were produced in spermatogonial cells after the administration of Aroclor 1242 at 1250 and 2500 mg/kg; the proportions of cells with abnormalities were 0,003 and 0.005, respectively, as compared to a mean of 0,004 in the control group (Table 4). Greater numbers of abnormalities were produced in the groups treated with 5000 and 500 mg/kg but the differences were not large enough to be statistically significant. The lesions produced were all of the single chromatid type. 1 I, >11111. QSW 026342 STLCOPCB4010304 Vi ' Discussion The results presented here clearly demonstrate the cumulative toxicity of Aroclor 1242 when administered in a multiple regimen. The 500 mg/kg dosage given acutely would have caused few if any deaths, as indicated by the results obtained with the 1250 mg/kg dosage (Table 1); however, when 500 mg/kg was given each day for 4 days, more than 507. of the animals died. Although the Aroclors are known to be relatively non-toxic in acute administration, we have found that when testing for possible mutagenic effects the usual assumptions do not always apply. Therefore both acute and multiple regimens were instituted in this investigation. When negative d8ta with respect to chromosomal abberations are presented utilizing compounds of this type, questions are raised concerning the -accessibility of the tissues to the compounds. The mitotic index serves as an Indicator, although not definitively, of the bioavailability of the compound under investigation. Generally, if inhibitlon of cell division is observed it is felt that the compound Or a breakdown product does penetrate the tissue in question. Ihi6 precludes a generalized toxic condition in the animal as a factor. Therefore, although chromosomal aberrations were not Been, the lack of such an effect was not considered to be a result of the inaccessibility of the tissue to the compounds. In addition, since these compounds are highly lipid-soluble, , the cell membrane would be expected to offer little resistance to their passage. Mitotic inhibition of bone marrow cells was completely absent with exception of the subacute dosage in the 6tudy of Aroclor 1242, and was barely evident in the study of Aroclor 1254. The inhibition produced by Aroclor 1254, although not very marked, may be due to a generalized tercic condition nt the highest dose levels; both levels produced comparable inhibition. The mean weight loss of these groups appeared to be dose-related and it had been expected that the inhibition would parallel this dose-response effect. With respect to spermatogonial cells, the inhibition produced by Aroclor 1242 appears to be genuine in that the da ta are not consistent with the view that a generalized toxic condition of the animals caused mitotic inhibition. If this were the etiology of the inhibition, then the single dose of 2500 mg/kg should have produced inhibition as well as the multiple doses of 500 mg/kg, since both produced approximately the 6ame degree of toxicity. The significance of the mitotic inhibition of spermatogonial cells is open to speculation. It was originally thought that mitotic inhibition could lead to reduction in the number of viable sperm in approximately 10 weeks, the amount of tisie required for spermatogonia to become mature sperm in the rat, and at that time the reduction could influence the reproductive performance of male animals. This 20 I I I I ' : : ' . I I i Jj I i ! ; , . ; ' , ; i ! [ ' DSW 026343 STLCOPCB4010305 I I In a would lultS ' I w 50% of be found isual i and o< ra- an billty b tbicompound cation. Inal as were 1 to be xnpounde. Luble, ilatance ely dy of odor h not n at e' red to it loo Q the i toxic this e of ie imatcly .totic n. It ' to ly 10 become could This k possibility has been investigated and it appears not to be the case (GRiEN( SAURO, and FRIEDMAN, nanuEcript in preparation). Two factors which may have prevented the manifestation of a reduction in reproductive performance are that ample numbers of spermatogonia were able to divide and become mature sperm at week 10, or that the inhibition of mitosis may have been concomitant with a slowing of the entire spermatogenic cycle, causing post-gonial cells to require 10 weeks or more for maturation. Hie investigation of the mutagenic potential of these com pounds through the cytogenetic examination of rat bone Diarrow and spermatogonia cells does not preclude the possibility that point mutations occurred, since they are not measurable in the procedure. There are, however, very few instances in which point mutations are known to occur without chromosomal aberrations occurring at a higher concentration in the same or in another system. In any case, on the this study, the Aroclors 1242 and 123^ possess no mutagenic potential as assessed by cytogeneticTnalysis of rat bone marrow and spermatogonia^ Acknowledgments Hie authors thank Mr. Kovel Davenport for valuable technical assistance, Ms. Linda Lofton for preparation of the manuscript and Ms. Janet A. Springer for Statistical evaluation of the data. References ALLEN, J.R., and D.H. NORBACK: Science .172. *98 (1973). ALVARES, A.P., D.R. BICKERS, and A. KAPPAS: Proc. Nat. Acad. Sci. U.S.A. 70, 1321 (1973). ANONYMOUS: Polychlorinated biphenyls and the environment, pp. 1-8. National Technical Information Service, U.S. Department of Commerce, Springfield, Va., 1972. BIROS, F.J., A.C. WALKER, and A. MEDBERY: Bull. Environ. Contain. Toxicol. 5, 317 (1970). BRUCKNER, J.V., K.L. KHANNA, and H.H. CORNISH: Toxicol. Appl. Pharmacol. 24, 434 (1973). EPSTEIN, S.S., E. ARNOLD, J. ANDREA, W. BASS, Bnd Y. BISHOP: Toxicol. Appl. PhanMcol. 22, 288 (1972). FISHBEIN, L.: In Polychlorinated biphenyls and the environment, pp. 124-131, National Technical Information Service, U. S. Department of Comnerce, Springfield, Va., 1972. Kwmn 21 QSW 026344 rTW STLCOPCB4010306 FRIEND, M., and D.O. TRAINER: Science L70, 1314 (1970>. HANSEN, D.J., P.R. PARRISH, J.I. LOWE, A.J. WILSON, JR., and P.D. WILSON: Bull. Environ. Contain. Toxicol. j>, 113 (1971). HOO. S.S., and C.A. BCWLES: Mutation Rea. n., 85 (1971). KEPLINGER, M.L., O.E. FANCHER, and J.C. CALANDRA: Toxicol. Appl. Piiarmacol, JJ), 402 (1971). KIMBROUGH, R.D.: Interagency Meeting on PCBs, U. S. Department of Health, Education, and Welfare, Washington, D.C., Aug. 5, 1971. KOLBYE, A.C.: Environ. Health Perspect. No. _1, 85 (1972). KURATSUNE, M., T. YOSHIMURA, J. MATSUZAKA, and A. YAMAGUCHI: Fukuoka Acta Med. 60(6), 513 (1969). KURATSUNE, M. , T. YOSHIMURA, J. MATSUZAKA, and A. YAMAGUCHI: Environ. Health Perspect. No. JL_, 119 (1972). LEGATOR, M.S., K. PALMER, S. GREEN, and K. PETERSEN: Science 105, 1139 (1969). LICHTENSTEIN, E.P., K.R. SCHULZ, T.W. FUHREMANN, and T.T. LIANG: J. Econ. Entomol. 62,, 761 (1969), / , LITTERST, C.L., AND E.P. LICHTENSTEIN: Arch. Environ. Health 22, 454 (1971). LITTERST, C.L., T.M. FARBER, A.M. BAKER, and E.J. VAN LOON: Toxicol. Appl. Pharmacol. 23, 112 (1972), MOSTELLER, R., and C. YOUTZ: Bioraetrika 48, 433 (1961). PLATONOW, N.S., R.M. LIPTRAP, and H.D. GEISS1NGER: Bull. Environ. Contain. Toxicol. ]_, 358 (1972). STANOVICK, R.P., S.I. SHAHIED, and E. MISSAGHI: Bull. Environ. Contain. Toxicol. JO, 101 (1973). TARRANT, K.R., and J.O.G. TATTON: Nature 219, 725 (1968). VEITH, G.D., and G.F. LEE: Water Res. 4, 265 (1970). ZITRO, V.: Bull. Environ. Contain. Toxicol. 2 105 (1972). DSW 026345 n STLCOPCB4010307