Document 7Z57ERZ5DdGbyyRk4m3gvqvR

EFFECT OF SIMULTANEOUS ADMINISTRATION OF POLYCHLORINATED BIPHENYLS AND ALKYLBENZENE SULFONIC ACID SALT IN RATS YOSHINORI ITOKAWA, M.D. Department of Hygiene, Faculty of Medicine Kyoto University, Kyoto, Japan RYO TABEl, M.D. ' Department of Pathology, Faculty of Medicine Kyoto University, Kyoto, Japan KAZUTAKA KaMOHARA, and K.UNISATO FUJIWaRA, PH.D. Kyoto City Institute ofPublic Health Kyoto, Japan Administration of PCB to rats resulted in an increase in liver weight, hypertrophy with foam-like changes in cytoplasm of individual liver cells, an increase in liver cholesterol levels, an increase in liver aniline hydroxylase activity and a decrease in liver Na-K-Mg-dependent ATPase activity. When ABS was simultaneously adminis tered with PCB, most of the above-mentioned abnormalities were observed to inCivdC ami vveic ucpcmleiil UII duuliimiiduuil iuuc auu uo^dge. Tcsucutat auiiuimalities were observed only in rats on long term co-administration of PCB and ABS. In 1968, over 1,000 persons in Kita Kyushu district (Southern Japan) suffered from blindness, gastrointestinal symptoms plus abdominal pain, jaundice, edema and a der matitis following ingestion of rice oil which contained 2,000 to 3,000 ppm of poly chlorinated biphenyls (PCB) (Kuratsune et at, 1969). Alkylbenzene sulfonic acid salt (ABS) is an industrially used neutral cleansing agent which has universally accumulated in the waterways. The LD50 of ABS is in the range of 500 to 3,000 mg/kg to various mammals (Swisher 1968). Chronic toxicity was examined by Bornmann (1963). Me administered ABS to the rats in the drinking water at 100 ppm for two years and no significant difference was observed between the ABS-fed animals and the controls. How ever, the toxicity of this substance is still highly debatable (Buehler et at. 1971, Yanagisawa 1963, Tokoro et al. 1964). As both PCB and ABS are ingested by humans, the authors attempted to discern the combined effect of these substances on rats, the results of which are outlined herein. Materials and methods Kancchlor 500 (a Japanese manufactured PCB containing as its main components pcntachlorobiphcnyi), was used. ArrKivn nf Is MQNS 085794 116 Y. Itokawa ct al. Reagent grade ABS (laurylbenzene sulfonic acid sodium salt, Wako Chemical Co., Japan) was employed. Male rats of the Wistar strain were housed individually in stainless steel cages with a raised wire bottom. Experiment No. I. Rats weighing about 60 g were divided into four groups of twelve animals each. To each group the following diets and water were given. Group 1: Group 2: Group 3: Group 4'. normal diet and tap water. normal diet and water containing 1,000 ppm of ABS. 500 ppm of PCB-supplemented diet and tap water. 500 ppm of PCB-supplemented diet and tap water containing^ ,000 ppm of ABS. Experiment No. II. Rats weighing about 150 g were divided into eight groups of eight animals each. To each group the following diet and water were given. Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7: Group 8: normal diet and tap water. Ten ppm of PCB-supplemented diet and tap water. 100 ppm of PCB-suppiemented diet and tap water. 500 ppm of PCB-supplemented diet and tap water. normal diet and water containing 1 ,000 ppm of ABS. Ten ppm of PCB-supplemented diet and water containing 1,000 ppm of ABS. 100 ppm of PCB-supplemented diet and water containing 1,000 ppm of ABS. 500 ppm of PCB-suppiemented diet and water containing 1,000 ppm of ABS. IXets and water were provided ad libitum for the duration of the experiment. Diets and water intakes were determined every two days and no significant differences in diet (average: 17.3 g/day) and water (average: 15.2 ml/day) intakes were observed between any of the groups. The composition of normal diet (Oriental Food Co.. Japan) is as follows (in 100 g diet): protein, 24.2 g; fat, 5.5 g; fiber, 4.5 g; carbohydrates, 52.6 g; salts, 6.2 g; water, 7.0 g; vitamin A, 1,000 I.l).; vitamin D, 200 I.U.; vitamin E, 1.6 mg; vitamin B,, 0.9 mg; vitamin B2, 0.8 mg; vitamin B6, 0.8 mg; niacin, 4 mg; pantothenic add, 1.9 mg; vitamin C, 50 mg; inositol, 60 mg; choline, 100 mg. Analytical Procedure. After a given period on these dietary regimens, blood specimens were obtained from the descending aorta using a plastic syringe. Serum was centrifugaily separated from blood clot, and brain, heart, liver, kidneys, spleen, and testicles were dissected and weighed. Portions of these tissues and serum were used for analysis. Serum alkaline phosphatase activity was assayed by the method of Hansen (1966), and choline esterase activity (Weber 1966), glutamate-oxaloacetate transaminase (GOT) MOWS 0B5795 Effect of PCD and ABS on Rats 117 and glutamate-pyruvate transaminase (GPT) activities (Reitman and Frankei 1957) were also determined. Cholesterol levels in serum were determined by the method of Abell et al. (1952), total and free cholesterol levels in liver by the method of Sperry and Webb (1950), serum and liver trigylccride levels according to Sardesai and Manning (1968), and seruin and liver non-estcriOcd fatty acids levels by the procedure of Duncombe (1964). Aniline hydroxylase activity in liver was assayed by the method of Imai et al. (1966) and Na-K-Mg-dcpendent ATPase and Mg-dcpendent ATPase by the method of Nakao et al. (1965). Iron levels in liver and scrum were determined by the bathophenanthroline method (Beale et al. 1961), serum urea by the method using diacetyimonoximclhioscmicarbazid (Coulombe and Favreau 1963), protein by the procedure of Lowry et al. (1951), and DNA and RNA levels in liver by the method of Ceriotti (1955). For histological analysis, all tissues were fixed in 3% formaldehyde solution. Sections of 5 u thickness were cut and stained with hematoxylin and eosin. Results Experiment No. I. After 1, 3 and 7 months on diets, four rats from each group were sacrificed and the body weights determined (Table 1). in the control group ana abSadministered group (groups 1 and 2) good growth was observed, while growth in the PCB-administered group and the PCB-ABS co-administercd group (groups 3 and 4) was depressed significantly. Actual liver weight, the weight of liver and testicles expressed as percentage of body weight for each group are shown in Table II. In PCB-administered groups 3 and 4, liver weight increased significantly; however in the ABS-administered group (group 2), liver weight was normal. Regarding actual liver weight, the weight was greater in group 4 than in group 3 at seven months. Fig. 1 shows the gross appearance of the Uvers. Light-microscopic examination of livers of groups 3 and 4 revealed swelling of the individual hepatic cells, nuclei showed picnosis, cytoplasm had many vacuoles and de generative changes were prominent in scattered areas of the liver. (Fig. 2A, normal liver; 2B, groups 3 and 4). DNA, RNA, and protein content and concentration in livers are shown in Table III. Liver DNA concentration was decreased in the PCB-administered groups (groups 3 and 4), however the total DNA content in liver underwent no significant change as liver weight was increased in the PCB administered groups. In contrast, total RNA and protein con tent per liver increased significantly in proportion to the increase of liver weight in PCBadministered rats. No change was observed in RNA and protein with respect to concentrations. HONS 0B5796 Group i 2 3 4 Table l Initial and terminal body weight of rats in expervnent / Additives to Diet NONE NONE PCB (500 ppm) PCB (500 ppm) Water NONE ABS (1000 ppm) NONE ABS (1000 ppm) Initial Weight (g) 62 + 5* 62 4* 63 6* 64 8* Terminal Weight (g)c 1 Month 216 + 7* 219 12* 185 J0*y 176 8* 3 Months 303 7* 263 23* 228 15> 231 13V 7 Months 371 +19* 351 10* 250 Il>' 245 9y Values represent mean S.E. t> 12 rats c4 rats average Data were considered statistically based on the method of one factor ANOVA. Different letter superscripts (x,y) following mean and S.E. denote significant differences (p < 0.05) between groups in each column, (eg, entries with superscript "x" showed no significant differences between groups; those with superscript "y" showed significant differences on comparison with groups not marked "y")- 1 MQNS 0 8 5 7 9 7 7c z c oe V *0 9 m Table IL ^c/wa/ /war weight, ratio ofliver and testicular weight to body weight in experiment / Group Additives to Diet Water . 1 Month Experimental period 3 Months 7 Months Actual Liver Weight (6) (6) (6) 1 NONE NONE 8.51 0.84* 8.64 0.51* 9.53 0.21* 2 NONE ABS (1000 ppm) 9.11 1.03* 8.57 0.27* 8.78 0.18* 3 PCB (500 ppm) NONE 13.73 t 1.77b 13.93 0.71 b 15.60 0.79b 4 PCB (500 ppm) ABS (1000 ppm) 13.83 16.33 2.12bc 19.25 2.1 5C Liver Weight/ Body Weight c%) 1 NONE NONE 3.94 0.41* 2 NONE ABS (1000 ppm) 4.16 0.32* 3 PCB (500 ppm) NONE 7.42 t i.) lb 4 PCB (500 ppm) ABS (1000 ppm) 7.86 1.23b +o Testicles Weight/ Body Weight 1 NONE NONE 2 NONE ABS (1000 ppm) 0.59 0.02* 3 PCB (500 ppm) NONE 0.61 0.04* 4 PCB (500 ppm) ABS (1000 ppm) 0.63 t 0.06* t Values represent mean S.E. of 4 rats. Different letter superscripts (a, b, c) denote significant difference (p < 0.05) in each tissue. Statistical analysis was earned out according to the method oi one factor AN'OVA. <%) 2.85 0.15* 3.26 0.46* 6.11 0.43b 7.07 1.08b 0.52 t 0.02* 0.58 0.04* 0.66 0.08* 0.63 0.06* . <%) 2.57 0.12* 2.50 0.30* 6.24 1 0-83b 7.86 1.50b 0.44 0.02* 0.46 0.01* 0.50 0.02* 0.26 0.03b o 3 03 n > Tn 3 = 120 Y. Itokawa el at. Table IIL DNA, RNA, and protein levels In liver (7 months) in experiment I DNA Group i 2 3 4 Additives to Diet Water . Concentration (/) NONE NONE PCB PCB NONE ABS NONE ABS 2.73 0.09* 2.65 * 0.16* 1.81 t0.ll1> 1.69 0.2I>> Content (ntg/Uvef) 26.45 4 1,31* 24.72 1.16* 28.15 4 0.89* 28.80 4 1.07* RNA Protein 1 2 3 4 1 2 3 4 NONE NONE PCB PCB NONE ABS NONE ABS NONE NONE PCB PCB NONE ABS NONE ABS 8.27 0.73* 7.95 t 0.90* 7.45 0.45* 7.78 4 0.40* <%) 19.5 2.0* 20.4 1.8* 18.8 2.8* 18.5 4 2.2* 79.9 4 2.6* 73.5 4 4.0* 117.0 4 11.b 138.8 4 19.2*> (g/liver) 1.8810.21* 1.88 4 016* 2.94 0.26b 3.3S 0.31 Values represent mean 4 S.E. of 4 rats. Different letter superscript* denote significant difference (p < O.OS) in each nucleic acid or protein. Statistical analysis was carried out according to the method of one factor ANOVA. Fig. 1. Grots appearance of liver of groups 1 to 4 (Experiment l. 7 months). Blackish color change and hypertrophy seen in PCB (group 3) and ABS + PCB treated rat (group 4). 0857^9 rtONS Effect of PCB end ABS on Rut 121 Testicle weight decreased in the individuals in group 4 after seven months treatment (Table 2). Histological observations of the testis of these rats revealed considerable de-. generation. Necrosis was observed in the seminiferous tubules (Fig. 3B), there was a disappearance of spermatogonium cells (Fig. 3B), hypertrophy of the interstitium between the tubules (Fig. 3B, 3C) and in some cases, the appearance of bizarre sperndogoniums (Fig. 3C). No significant changes in tissue weight or histological appearance were observed in other tissues, A Fig. 2. Microscopic appearance of liven. (Experiment I, 7 months) Hsemitoxylin-eosin, X100. A. Normsi rat (group !). Nuclei and cytoplasms in ill hepatocytes art normal. B. PCB-ABS treated rat (group 4). Note foamy degeneration in many hepatocytes. 0B5o Iiokawa f t (' TO BSPO SNOW Fig. 3. Microscopic appearance oftestis. (Experiment I, 7 months), HaematoxyJin-eosin, X100. A. Normal rat (group 1). The seminiferous tubules and interstitial tissues arc normal in appearance. B. PCB-ABS treated tat (group 4). Note empty tubules and hyperplasia of the interstitial tissues. C. PCB-ABS treated rat (group 4). Note appearance of bizarre sperniogoniums as well as hyperplasia of the interstitial tissue. Effect of PCB and ABS on Rats 123 GOT and GPT levels in the serum of groups 3 and 4 (seven months) increased (Table IV). There was no signiflcant change in serum alkaline phosphatase and choline esterase activities. Cholesterol levels in the serum increased in groups 3 and 4 alter 1. 3 and 7 months treatment (Table V). Total cholesterol levels in the liver increased markedly In group 3 and particularly in group 4 after 3 and 7 months. Liver total cholesterol levels increased with administration time. (Table V). Liver aniline hydroxylase activity increased significantly (p < 0.01) in rats on a PCB-implemented diet, the increase being sugumented (p < 0.01) when PCB and ABS were combined. (Table VI). Uver Na-K-Mg-dependent ATPase decreased in both groups 3 and 4; however, decrease were greater in group 4. (Table VI). No significant difference was observed in Mg-dependent ATPase among the poups. Experiment No. II. As the above experiment indicated that the effect of PCB increased when ABS was simultaneously administered, a further study was done to determine the dose response. Growth was depressed only in groups 4 and 8 (administered 500 ppm of PCB); there was no significant change in growth among the other groups. The rats were sacrificed after one month and analysts revealed that the weights of the liven increased with in creased doses of PCB. Regarding Uver weight, a synergistic effect of ABS upon PCB was observed between groups 4 and 8 (500 ppm of PCB) only. (Table Vll). Serum urea levels increased in groups 4 and 8 (500 ppm of PCB). Iron levels increased In the groups 7 and 8. (Table VIII). Serum cholesterol and Uver free cholesterol levels were increased In group 8 only. Total cholesterol levels in the liver increased in the 100 and 500 ppm PCB groups 3,4,7 and 8. Cholesterol deposits were more marked in the PCB-ABS combined groups. (Table IX). Aniline hydroxylase activity increased and Ne-K-Mg-dependent ATPase decreased in proportion to the PCB administered. Mg-dependent ATPase was slightly decreased in the poups on 500 ppm of PCB. (Table X). There was no significant change in the triglyceride and non-esterified fatty acids levels in serum and liver. Dbcuflion Monkeys which had been administered 1.4 to 16 mg/day of PCB (Kanechlor 400) for 40 to 48 days revealed a Uver cell enlargement and fatty degeneration (Nbhizumi 1970), A similar morphological change in rat Uver was reported by Kimbrough et at. (1972> The present study demonstrated that the increase in rise and weight of the liver In the PCB groups is attributed to the enlargement of individual Uver ceUs. This con clusion was readied both from morphological examinations and chemical analysis of Uver DNA, RNA, and protein contents (Table III). Enhancement of the Uver drug metabolizing system by PCB administration has been reported by Fujita et *1. (1971), MONS 005802 I '. Itokawa et at. MGNS 0 8 5 8 0 3 Enzyme GOT .. GPT Table IV. Glutamete-oxaloacetate transaminase (GOT) end glutamate-pynrvate transaminase activities (GPT) in serum in experiment / (International Units) Group Additives to Diet Water 1 Month Experimental period 3 Months 7 Months 1 NONE NONE 17.9 1.2* 2 NONE ABS (1000 ppm) 18.2 1.7* 3 PCB (500 ppm) NONE 15.5 3.6* 4 PCB (500 ppm) ABS (1000 ppm) 16.1 2.7* 20.6 5.1* 18.3 5.0* 19.8 3.3* 22.7 4.9* 18.7 + 1.8* 23.1 4.2* 52.5 7.5b 54.2 7.4b 1 NONE NONE 24.1 0.7*b 2 NONE ABS (1000 ppm) 24.0 2.7*b 3 PCB (500 ppm) NONE 21.8 3.7ab 4 PCB (500 ppm) ABS (1000 ppm) 21.2 1.2* 21.8 5. l*b 20.9 1.2* 31.8 t 4.0ba 36.6 6.4bc 22.7 t 2.4*b 24.1 2.2*b 32.0 3.`K 44.2 t 7.5C Values represent mean S.E. of 4 rats. Different letter superscripts (a.b.c) denote significant differences (p < 0.05) in each enzyme. Statistical analysis was carried out according to the method of one factor ANOVA. Effect o f PCB and ABS on Rais Group Serum 1 2 3 4 liver 1 2 3 4 Table V. Total cholesterol levels in liver and serum in experiment I Additives to Diet Water 1 Month Experimental period 3 Months 7 Months NONE NONE PCB (500 ppm) PCB (500 ppm) NONE ABS (1000 ppm) NONE ABS (1000 ppm) NONE NONE PCB (500 ppm) PCB (500 ppm) NONE ABS (1000 ppm) NONE ABS (1000 ppm) (mg/100 ml) 106 13* 138 t 28ab 196 9bc 262 24* 87 7* 124 15* 210 19b* 220 24bc 94 4* 105 15* 211 19bc 230 15* (mg/100 g wet tissue weight) 241 17* 281 21 323 33b 389 + 74b* 255 31* 25,3 t 254 439 32bc 611 47d* 257 + 22* 320 +33* 548 344 768 i 67* ^O B S B O SNOW Values represent mean t S.E of 4 rats. . Different tetter superscripts (a.b.c^.e) denote significant differences, (p ^ 0.05) in sctuui oi liver. Data were considcted statistically based on the method of one factor ANOVA. 12J. f l . Jlokawa ct al. Table VI Aniline hydroxylase, Na-K-Mg-dependent ATPase and Mg-dependent ATPase in liver in experiment I Enzyme Group Diet Additives to Water Experimental period 3 Months 7 Months Aniline Hydroxylase Na-K-Mg-ATPase i 2 3 4 i 2 3 4 NONE NONE PCB (500 ppm) PCB (500 ppm) NONE ABS (1000 ppm) NONE ABS (1000 ppm) NONE NONE PCB (500 ppm) PCB (500 ppm) NONE ABS (1000 ppm) NONE ABS (1000 ppm) (m^imoles p-aminophenol/mg prot./min) 1.1 6 0.06" 1.42 0.12a 3.94 0.38b 6.72 0.571= 1.48 0.21a 1.22 0.14> 4.19 0.46 6.90 0.66= (m^imoles Pi/mg prot./min) 140 7J 122 21 78 St> ' 45 12C 155 52 130 7a 76 6b 44 10c Mg-ATPase 1 NONE NONE 401 +34 2 NONE ABS (1000 ppm) 424 32a 3 PCB (500 ppm) NONE 37 313 4 PCB (500 ppm) ABS (1000 ppm) 343 t 29a 328 + 47* 329 + 56a 34 2 1 35a 399 85a Values are represent mean S.E. of 4 rats. Different letter superscripts following mean and S.E. (a.b.c) denote significant differences (p < 0.05) in each enzyme. Data were considered statistically based on the method of one factor ANOVA. 5 0 S S B 0 SNOW Effect of PCB and ABS on Rats 127 Table VII Ratio of Inter weight to body weight (one month) in experiment II Croup Diet Additives to Water % 1 NONE NONE 2.94 0.06* 2 PCB (10 ppm) NONE 3.01 t 0.05*b 3 PCB (100 ppm) NONE 3.93 0.14 4 PCB (500 ppm) NONE 5.82 * 0.50d 5 NONE ABS (1000 ppm) 2.92 0.07* 6 PCB (10 ppm) ABS (1000 ppm) 3.12 0.06*> 7 PCB (100 ppm) ABS (1000 ppm) 4.05 0.14c 8 PCB (500 ppm) ABS (1000 ppm) 6.45 0.24* Values represent mean S.E. of 8 rats. Different letter superscripts following mean and S.E. (a,b,c,d,e) denote significant differences (p < 0.0S) compared by the method of one factor ANOVA. Table VIU Urea and iron levels in serum (one month) in experiment II Group Additives to Diet Water Urea (mg/100 ml) Iron (jig/tOO ml) 1 NONE NONE 20.9 0.8a 157 18* 2 PCB (10 ppm) NONE 22.8 2.1` 149 i 16 3 PCB (100 ppm) NONE 22.2 1.5*b 180 I7b 4 PCB (500 ppm) NONE 27.4 2.3 1761 21b 5 NONE ABS (1000 ppm) 22.8 2.0*bc 198 36a* 6 PCB (10 ppm) ABS (1000 ppm) 23.7 2.5lbc 206 43*> 7 PCB (100 ppm) ABS (1000 ppm) 23.5 t 2.0*b* 2161 15*b S PCB (500 ppm) ABS (1000 ppm) 28.8 t 2.9*4 236 1 31" Values represent mean * S.E. of 8 rats. Different letter superscripts following mean and S.E. (a.b.c.d) denote significant differences (p < 0.0S) compared by the method of one factor ANOVA. MONS 085806 121 Y. Itofcawaet a/. Table IX Total cholesterol levels in serum and total and free cholesterol levels in liver /one month) in experiment II Croup Additives to Dill Water Serum (mg/100 mi) Liver Total Cholesterol Free Cholesterol (mg/100 g wet weight) 1 NONE NONE 109 t 17 290 14* 214 1 10* 2 PCB (10 ppm) NONE 104 15* 2S9 i 18*` 2191 16* 3 PCB (100 ppm) NONE 113 20* 336 23be 224 1 12* 4 PCB (500 ppm) NONE 1411 28>b 421 t 254 254 1 30*b 5 NONE ABS(IOOOppm) 981 16* 268 1 19* 225 1 14* 6 PCB (10 ppm) ABS (1000 ppm) 116 26* 265 16* 237 1 15* 7 PCB(lOOppm) ABS (1000 ppm) 123 23* 354 t 31*4 225 1 10* S PCB (500 ppm) ABS (1000 ppm) 180 13* 522 15* 313 26b Values represent mean i S.E. of S rata. Different letter superscripts following mean and S.E. (a, b, c. d, e) denote rignmuaul differences in each column (p < 0.0S) compared by the method of one factor ANOVA. VUeneuve et at. (1971) and Litterst et a/. (1972). Matsuds (1959) and Tanaka et al. (1969) observed that total lipid, triglyceride and cholesterol contents in the liver in creased when PCB was ingested. Inhibition of ATFue activity in fish has been reported by Yap et al. (1971). The present studies confirmed the various abnormalities attributed to PCB administration and proved that some of them are increased by simultaneous feeding of PCB and ABS. It is noteworthy that PCB inhibited the Na-K-Mg-depenflent ATPase not only in a cold-blooded animal but also in mammals. In various analyses of die serum, increase of transaminase observed in the high-dosage PCB groups (seven months) could be due to degenerative changes in the liver. The hemolytic effect of ABS could explain the increase of the serum iron levels in the ABS-administered groups. It is of interest to note the morphological testicular abnormalities observed in ABS-PCB co administered rati (seven months). With regard to the effect of PCB as a mitotic poison. FbakaD et al. (1972) reported that frequencies of chromosome aberrations increased in PCB-treated dove embryos as compared to control embryos. The following mechanisms may be considered for the etiology of the observed testicular atrophy: (1) PCB disturbs primarily the testicular vasculature, (2) PCB affects genetic materials as a mitotic poison. (3) PCB causes a nutritional deficiency, such is Vitamin E deficiency, (4) PCB affects certain enzyme activities in the testicles, (3)PCB affects the endocrine control of testicular integrity, and (6) these effects could be caused by an enhanced absorption of PCB ss a MONS 065007 Table X Aniline hydroxylase, Na-K-Mg-dcpendent ATPase andMg-dependent ATPase activities in liver (one month) in experiment II Effect o f PCB and ABS on Rats Croup Diet Additives to Vfiter 1 NONE 2 PCB (10 ppm) 3 PCB (100 ppm) 4 PCB (500 ppm) S NONE 6 PCB (10 ppm) 7 PCB {100 ppm) S PCB (500 ppm) NONE NONE NONE NONE ABS (1000 ppm) ABS (1000 ppm) ABS (1000 ppm) ABS (1000 ppin) Aniline hydroxylase (m moles /mg prot./min) 1.21 0.16* 1.76 0.23* 3.96 0.48" 5.00 0.62" 1.63 0.13* 1.81 0.19* 4.15 0.41" 5.51 0.66" Na K-Mj ATPase Mg-ATPase (m moles/mg prot./min) 117 I6*b 113 20*"* 80 19"* 60 20*" 135 21* 99 17*"* 81 12"* 42 5" 306 59*" 311 37* 265 39*" 251 22*" 336 48* 307 55*" 300 27* 226 20" B 0 8 S B 0 SNOW Vaf'ferei'kX'm',,`* S E 0f 8 r>ts ' ', one factor ANOVa"*'"1'15 denote significant differences (p < 0.05) in each column compared by the method of 130 Y. hokawa et at. result of the emulsifying action of ABS. Although further experiments are necessary to elucidate the mechanism, attention is drawn to the effects of PCB (or PCB plus ABS) on the reproductive physiology of male rats. Acknowledgments The authors express their gratitude to Professor M. Fujiwaia, Department of Hygiene, Faculty of Medicine, Kyoto University and to Dr. T. Tads, Head of Kyoto City Institute of Public Health for support of this work. Thanks are also due to Misses Y. Natori and Uang-Fang Tseng for technical assistance. References Abell, L L, B. B. Levy, B. B. Brodie, and K. Kendall: A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J. Biol. Chem. 195,357(1952). Beale, R. N., J. 0. Bostrom, and R. F. Taylor: Rapid incremental methods for the deter mination of serum iron and iron binding capacity. J. Clin. Pathol. 14,488 (1961). Bornmann, G., and A Loeser: The biological effects of dodecylbenzene sulfonate sur factants on rats. Fette Seifen Anstrichmittel 65, 814 (1963). Buchlcr, E. V., E. A. Ncwinaim. and W. R. King: Two-year feeding and reproduction study in rats with linear alkylbenxene sulfonate (LAS). Toxicol. Appl. Pharmacol. 18,83(1971). Ceriotti, G.: Determination of nucleic acids In animal tissues. J. Biol. Chem. 214, 59 (1955). Coulombe, J. J., and L. Fsvreau: A new simple semimicro method for colorimetric determination of urea. Clin. Chem. 9,102 (1963). Duncombe, W. G.: The colorimetric micro-determination of non-esterifted fatty acids in plasma. Clin. Chim. Acta 9,122 (1964). Fujita, S., H. Tsuji, K, Kalo, S. Saeki, and H. Tsukamoto: Effect of biphenyl chlorides on tat liver microtomes. Fukuoka Acta Medica 62, 30 (1971). Hansen, P. W.: A simplification of King and King's method for determination of serum phosphatase. Scand. J. Clin. Lab. Invest. 18, 353 (1966). bnai, Y., A. Ito, and R. Sato: Evidence for biochemically different types of vesicles in the hepatic microsomal fraction. J. Biochem. 60, 417 (1966). Kimbrough, R. D., R. E Under, and T. B. Gaines: Morphological changes in livers of rats fed polychlorinated biphenyls. Arch. Environ. Health 25,354 (1972). Kurataune, M., T. Yoshimura, J. Matsuzaka, and A Yamaguchi: Yusho, a poisoning caused by rice oil contaminated with chlorobiphenyls. Fukuoka Acta Medica 60, 513(1969). . IP MQNS 065809 ts are necessary to t PCB plus ABS) on apartment of Hygiene, h Kyoto City institute I Misses Y. Natori and llfled method Tor the ill specificity. J. Biol. nethods for the deter101.14,488(1961). icnzene sulfonate sor ing and reproduction ;ol. Appl. Pharmacol. Biol. Chcm. 214, 59 Ihod for colorimetric Aerified fatty acids in biphenyl chlorides on teimination of serum ypes of vesicles in the tinges in liven of rats (1972). : Yusho, a poisoning loka Acta Medlca 60, Effect of PCB and ABS on Rais 131 Litterst, C. J., T. M. Farber, A. M. Baker, and E. J. Van Loon: Effect of polychlorinated biphenyls on hepatic microsomal enzymes in the rat. Toxicol. Appl. Pharmacol. 23, 112(1972). Lowry, O. H., N. J. Roscbrough, A. C. Farr, and R. J. Randall: Protein measurement with the folin-pheno! reagent. J. Biol. Chcm. 193, 265 (1951). Matsuda, T Hie experimental studies of effect of polychlorinated biphenyl on lipid metabolism. Kokumin Eisei 28, 672 (1959). Nakao, T., Y. Tashima, K. Nagano, and M. Nakao: Highly specific sodium-potassiumactivated adenosine triphosphatase from various tissues of rabbit. Biochcm. Biophys. Res. Comm. 19,755 (1965). Nishizumi, M: Light and electron microscope study of chlorobiphenyl poisoning. Arch. Environ. Health 21, 620 (1970). Peakall, D. B,, J. L Lincer, and S. E. Bloom: Embryonic mortality and chromosomal alterations caused by Aroclor 1254 in ring doves. Environ. Health Perspectives 1. 103 (1972). Rcitman, S., and S. Frankel: A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminase;. Amer. J. Gin. Pathol. 28, 56(1957). Sardesai, V. M., and J. A. Manning: The determination of trigylcerides in plasma and tissues. Gin. Chem. 14, 156(1968). Sperry, W. M., and M. Webb: A revision of the Schoenheimer-Sperry method for cholesterol determination. J. Biol. Chem. 187,97 (1950). Swisher, R. D.: Exposure levels and oral toxicity of surfactants. Arch. Environ. Health 17, 232(1968). Tanaka, K., S. Fujita, F. Komatsu, and N. Tamura: Experimental subacute poisoning of chlorobiphenyls particularly the influence on the serum lipids in rats. Fukuoka Acta Medica 60, 544(1969). Tokoro, Y., M. Hirasawa, and S. Moteki: Experimental histopathologic investigation upon the biological effect of alkylbenzene sulfonate in the long term intake. Jap. J. Public Health 11, 29 (1964). Villeneuve, D. C,, D. C. Grant, W. E. J. Phillips, M. L. Dark, and D. G. Gegg: Effects of PCB administration on microsomal enzyme activity in pregnant rabbits. Bull. Env. Contam. Toxicol. 6, 120(1971). Wfeber, H.: Rashe und einfache Ultramikromethode zur Bestimung dcr Serumcholinesterase. Dtsch med. Wschr. 91,1927 (1966). Yanagliawa, F.: On the neutral cleansing agent. Jap. J. Public Health 10,413 (1963). Yap, H. 1L, D. Desaiah, L. K. Cutkomp, and R. B. Koch: Sensitivity of fish ATPase to polychlorinated biphenyls. Nature 233, 61 (1971). Manuscript received January 29, 1974; accepted May 4, 1974 MGNS 085810