rOXICOLOGY
AND
APPLIED
Toxicological III. Comparison
N.
BICHET,
D. Sanoj
106, 509-517
PHARMACOLOGY
CAHARD,
(1990)
Studies on a Benzofuran
Derivative
of Peroxisome Proliferation in Rat and Human Hepatocytes in Primary Culture
G.
FABRE, B. REMANDET, D.
GOUY,
AND
J-P.
CANO
Recherche, 371 rue du Pr J. Blayac, 34184 Montpellier, France
Received November 2, 1989; accepted July 27, 1990 Toxicological Studies on a Benzofuran Derivative. III. Comparison of Peroxisome Proliferation in Rat and Human Hepatocytes in Primary Culture. BICHET, N., CAHARD, D., FABRE, G., REMANDET, B., Gow, D., AND CANO, J-P. (1990). Toxicol. Appl. Pharmacol. 106,509-5 17. Primary cultures of rat and human hepatocytes were used in our in vitro studies for investigating species differences in the response to a peroxisome proliferating benzofuran derivative, benzbromarone. Cyanide-insensitive palmitoyl coenzyme A oxidation (a marker of peroxisome fatty acid p-oxidation) and electron microscopy were used to assess peroxisome proliferation. Hepatocytes were cultured essentially as described by Mitchell et al. (1984, Arch. Toxicol. 55, 239-246); clofibric acid and mono(2-ethylhexyl) phthalate (MEHP) were used as reference compounds, as they are well known to cause peroxisome proliferation in rat hepatocytes in primary culture. The benzofuran derivative, tested at drug concentrations ranging from 2.37 to 59.20 pM in rat hepatocyte primary cultures, induced, after 96 hr. a dose-related increase of the peroxisomal/3-oxidase activity correlated with an increased number of peroxisomes; this increase was much less marked than that obtained with clofibric acid or MEHP. By contrast, using the same range of concentrations, human hepatocytes in primary culture treated with benzbromarone revealed no enhancement of enzymatic activity and no concomitant statistically significant increase in the number of peroxisomes; the same observations were reported with clofibric acid and MEHP. These results demonstrate clearly that species differences in sensitivity to peroxisome proliferation with the benzofuran derivative do exist. 0 1990 Academic Press, Inc.
A variety of chemicals, including hypolipidemic drugs and plasticizers, are known to induce peroxisome proliferation in the liver of rodents and someother speciesincluding cats, dogs, chickens, and sometimes in some nonhuman primates (Reddy et al., 1984; Gray and De La Iglesia, 1984). These agentshave shown marked species differences in their effects (Reddy and Lalwani, 1983; Reddy et al., 1984; Reddy and Rao, 1987; Elcombe and Mitchell, 1986) and even strain differences in mice (Butler et al., 1988; Dwivedi et al., 1989). Certain speciessuch as guinea pigs, marmosets, and rhesus monkeys have displayed little re509
sponseor even an absenceof the induction of hepatic peroxisomes (Rodricks and Tunnbull, 1987; Lake et al., 1989). In particular, induction of peroxisomes in humans is equivocal (Hanefeld et al., 1983; Gariot et al., 1987). The increase in the number of peroxisomes and the induction of associatedenzyme activities (e.g., catalaseand oxidases)have alsobeen demonstrated in vitro in primary cultures of rodent hepatocytes (Gray et al., 1983; Reddy and Rao, 1987; Butler et al., 1988; Furukawa et al., 1988), but never in cultured human hepatocytes (Mitchell et al., 1985; Elcombe and Mitchell, 1986). 0041-008X/90
$3.00
Copylight 0 1990 by Academic Press. Inc. ALI rights of reproduction in any form reserved.
510
BICHET
In the preceding papers, a benzofuran derivative, benzbromarone, was found to produce hepatic peroxisome proliferation in rats in viva (Butler et al., 1990). Therefore, the present study was undertaken to determine whether benzbromarone would do the same in cultured rat and human hepatocytes. We report here that rat hepatocytes in primary culture exposed to benzbromarone or the reference compounds, clofibric acid and mono(2ethelhexyl)phthalate (MEHP), displayed peroxisome proliferation whereas human hepatocytes, although metabolically competent, were refractory to peroxisome proliferation. MATERIALS
AND
METHODS
.Materials Clofibric acid, insulin. hydrocortisone, palmitoyl-CoA, amino-2-ethyl1,3-propanediol, and 3,3’-diaminobenzidine (DAB) were purchased from Sigma Chemical Co. (St. Louis, MO). MEHP was supplied by Elf Aquitaine ATOCHEM Division (Paris, France). The benzofuran derivative, benzbromarone, was obtained from Sanofi Recherche (Montpellier. France). Potassium cyanide (KCN) and hydrogen peroxide (H,O,) were obtained from Prolabo (Paris, France). 3-Amino1,2,4-triazole was obtained from Fluka (Switzerland). Leibowitz L15 culture medium and glutamine were supplied by GIBCO/BRL (Paris, France); streptomycin, penicillin, and collagenase by Boehringer SA (Manheim, France). Tryptose phosphate broth was obtained from FIow Laboratories SA (France), and the fetal calf serum was supplied by IBF (France). [li4C]palmitoyl-CoA (56 mCi/mmol) was obtained from Amersham. All other chemicals were of the highest available purity and were supplied by Sigma Chemical Co., Boehringer SA, or Merck (Darmstadt, FRG).
Hepatocyte
Isolation
and Culture
Rat hepatocytes were isolated from male SpragueDawley rats by a two-step in situ perfusion technique using collagenase as described by Williams et a/. (1982). The isolated cells were washed twice by centrifugation at 5Og and resuspended in Williams’ medium E supplemented with 10% newborn calf serum + streptomycin and penicillin (WMES). Cell viability, determined by the trypan blue exclusion test, ranged between 90 and 95%. Human hepatocytes were prepared from resected liver lobe from three patients with secondary liver cancer, two of them with colon adenocarcinoma, and the third with
ET
AL.
rectum adenocarcinoma. Lobes were perfused via a branch of the portal vein with a Hepes buffer containing collagenase, according to the method developed by Fabre et a/. (1988) on whole human livers and subsequently adapted for human liver lobes (Cano et al., 1989). The viability of hepatocytes. assessed by the trypan blue exclusion test and by a light microscopic morphological analysis, ranged between 80 and 95%. The functional viability of human hepatocyte cultures from each hepatocyte preparation was assessed by measurement of the inducibility of cytochrome P450 IA-mediated 7-ethoxyresorufin-O-deethylase activity after a 72hr exposure to /3-naphthoflavone (50 @M) and 3-methylcholanthrene (50 FM), and by the increase of the specific cytochrome P450 IIIA isozyme “nifedipine oxidase” activity by rifampicin (50 FM) and dexamethazone (50 PM) (Cano et al., 1989).
Treatment eration
of Hepatocyte
Cultures
for Peroxisome
Pro&f:
Rat and/or human cellular suspensions were immediately seeded at 2.5 X lo6 viable cells per 3 ml culture medium (WMES) in 60-mm petri dishes coated with collagen and maintained at 37°C in a humidified atmosphere (5% CO2 and 85% air). Three hours after seeding for rat preparations, and 7 to 16 hr after seeding for human preparations, the culture medium and unattached cells were aspirated and replaced by fresh L15 Leibowitz medium supplemented with 10% fetal calf serum, 10e6 M insulin, 10m5 M hydrocortisone, tryptose broth phosphate (8.3%) according to Mitchell et a/. (1984). Further medium changes were performed 24, 48, and 72 hr after seeding. For each treatment, four culture dishes were used, two for enzymatic activity assay and two for morphological analysis. The test compounds, clofibric acid and MEHP, to be added to the monolayers were dissolved in dimethylfonnamide (DMF); benzbromarone was dissolved in dimethyl sulfoxide (DMSO). The concentrations used were 0.1,0.5, 0.75, I, and 2 mM for clofibric acid and MEHP. Benzbromarone was tested at 2.37, 11.85, 23.70,47.40. 59.20, and 7 1.10 @M. Test compounds were added at every 24hr medium change, unless indicated otherwise. Addition of DMF or DMSO never exceeded 10 ~1 per petri dish (this concentration (j (J. M. Lowenstein, Ed.), Vol. 72, Part D, pp. 3 15-3 17. Academic Press, San Diego. LAZAROW, P. B. (1987). Rat liver peroxisomes catalyse the P-oxidation of fatty acids. J. Biol. Chern. 253, 15221528. LOWRY, 0. H., ROSEBROUCH,N. J., FARR, A. L., AND RANDALL. R. J. (195 1). Protein measurement with the Folin-phenol agent. J Bioi. Chem. 193, 265-275. MARSMAN, D. S.. CA~LEY, R. C., CONWAY, J. G., AND POPP, J. A. (1988). Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferators di(2ethylhexyl)phthalate and 4-chloro-6-(2,3-xylidino)-2pyrimidinylthio-acetic acid (WY-14,643) in rats. Cancer Res. 48,6739-6744. MITCHELL, A. M., BRIDGES,J. W., AND ELCOMBE, C. R. (1984). Factors influencing peroxisome proliferation in cultured rat hepatocytes. Arch. Toxicol. 55, 239-246. MITCHELL, A. M., JOSEPH, K., AND ELCOMBE. C. R. (1985). Species differences in hepatic peroxisome proliferation. Hum. Toxicol. 4, 552. NOVIKOFF, A. B., AND GOLDFISCHER,S. (1969). Visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine. J. Histochem. Cytochem. 17, 675-680.
NOVIKOFF, A. B., NOVIKOFF, P. M., DAVIS, C., AND QUINTANA, N. (1972). Studies on microperoxisomes. II. A cytochemical method for light and electron microscopy. J. Histochem. Cytochem. 20, 1006-1023. NOVIKOFF, P. M., NOVIKOE, A. B., QUINTANA, N., AND DAVIS, C. (1973). Studies on microperoxisomes. III. Observations on human and rat hepatocytea J. Histo&em. Cytochem. 21, 540-558. PARZEFALL, W., SCHUPPLER,J., BARTHEL, G., MEYERROZZE, B., AND SCHULTE-HERMANN, R. (1990). Toxicological studies on a benzofuran derivative. I. A comparative study with phenobarbital on rat liver. Toxicol. Appl. Pharmacol. 106, 482-499. RAO, M. S., AND REDDY. J. K. (1989). The relevance of peroxisome proliferation and cell proliferation in peroxisome proliferator-induced hepatocarcinogenesis. Drug Metab. Rev. 21, 102-l 10.
IN RAT AND HUMAN
LIVER
517
REDDY, J. K., AZARNOFF, D. L., AND HIGNITE, C. E. ( 1980). Hypolipidemic hepatic peroxisome proliferators form a novel “class” of chemical carcinogens. Nature (London) 283,397-398. REDDY,J. K., AND LALWANI. N. D. (1983). Carcinogenesis by hepatic peroxisome proliferators: Evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. Toxicol. 12, l-58. REDDY, J. K.. LALWANI, N. D., QURESHI, S. A., REDDY, M. K., AND MOEHLE, C. M. (1984). Induction of hepatic peroxisome proliferation in non-rodent species, including primates. Amer. J. Pathol. 114, 171-183. REDDY,3. K.. AND RAO, M. S. (1987). Xenobiotic-induced peroxisome proliferation: Role of tissue specificity and species differences in response in the evaluation of the implications for human health. Arch. Toxicol. Suppl. 10,43-53.
RODRICKS.J. V., AND TUNNBULL, D. (1987). Interspecies differences in peroxisomes and peroxisome proliferation. Toxicol. Ind. Health 3, 197-212. SCHULTE-HERMANN, R., OHDE, G., SCHUPPLER.J., AND TIMMERMANN-TROSIENER, I. (198 1). Enhanced proliferation of putative preneoplastic cells in rat liver following treatment with the tumor promoters phenobarbital, hexachlorocyclohexane, steroid compounds and nafenopin. Cancer Res. 41,2556-2562. SCHULTE-HERMAW, R. (1985). Tumor-promotion in the liver. Arch. Toxicol. 57, 147-158. STERNLIEB, I.. AND QUINTANA, N. (1977). The peroxisomes ofhuman hepatocytes. Lab. Invest 36, 140-149. 140-149. STOTT. W. T. (1988). Chemically induced proliferation of peroxisomes: Implications for risk assessment. Regul. Toxicol. Pharmacol. 8, 125-l 59. WILLIAMS, G. M., LASPIA, M. F., AND DUNKEL, V. C. (1982). Reliability of the hepatocyte primary culture/ DNA repair test in testing of coded carcinogens and noncarcinogens. Mutat. Res. 97, 369-370. WILLIAMS, G. M., AND WEISBURGER, J. H. (1986). Chemical carcinogens. In Toxicology. The Basic Sciences ofPoison (C. Klaassen, M. Amdur, and J. Doull, Eds.), 3rd ed., pp. 99-173. McMillan Co., New York. WILLIAMS, G. M., MARUYAMA, H., AND TANAKA, T. (1987). Lack of rapid initiating, promoting or sequential syncarcinogenic effects of di(2-ethylhexyl)phthalate in rat liver carcinogenesis. Carcinogenesis 8, 875-880.
AND
APPLIED
Toxicological III. Comparison
N.
BICHET,
D. Sanoj
106, 509-517
PHARMACOLOGY
CAHARD,
(1990)
Studies on a Benzofuran
Derivative
of Peroxisome Proliferation in Rat and Human Hepatocytes in Primary Culture
G.
FABRE, B. REMANDET, D.
GOUY,
AND
J-P.
CANO
Recherche, 371 rue du Pr J. Blayac, 34184 Montpellier, France
Received November 2, 1989; accepted July 27, 1990 Toxicological Studies on a Benzofuran Derivative. III. Comparison of Peroxisome Proliferation in Rat and Human Hepatocytes in Primary Culture. BICHET, N., CAHARD, D., FABRE, G., REMANDET, B., Gow, D., AND CANO, J-P. (1990). Toxicol. Appl. Pharmacol. 106,509-5 17. Primary cultures of rat and human hepatocytes were used in our in vitro studies for investigating species differences in the response to a peroxisome proliferating benzofuran derivative, benzbromarone. Cyanide-insensitive palmitoyl coenzyme A oxidation (a marker of peroxisome fatty acid p-oxidation) and electron microscopy were used to assess peroxisome proliferation. Hepatocytes were cultured essentially as described by Mitchell et al. (1984, Arch. Toxicol. 55, 239-246); clofibric acid and mono(2-ethylhexyl) phthalate (MEHP) were used as reference compounds, as they are well known to cause peroxisome proliferation in rat hepatocytes in primary culture. The benzofuran derivative, tested at drug concentrations ranging from 2.37 to 59.20 pM in rat hepatocyte primary cultures, induced, after 96 hr. a dose-related increase of the peroxisomal/3-oxidase activity correlated with an increased number of peroxisomes; this increase was much less marked than that obtained with clofibric acid or MEHP. By contrast, using the same range of concentrations, human hepatocytes in primary culture treated with benzbromarone revealed no enhancement of enzymatic activity and no concomitant statistically significant increase in the number of peroxisomes; the same observations were reported with clofibric acid and MEHP. These results demonstrate clearly that species differences in sensitivity to peroxisome proliferation with the benzofuran derivative do exist. 0 1990 Academic Press, Inc.
A variety of chemicals, including hypolipidemic drugs and plasticizers, are known to induce peroxisome proliferation in the liver of rodents and someother speciesincluding cats, dogs, chickens, and sometimes in some nonhuman primates (Reddy et al., 1984; Gray and De La Iglesia, 1984). These agentshave shown marked species differences in their effects (Reddy and Lalwani, 1983; Reddy et al., 1984; Reddy and Rao, 1987; Elcombe and Mitchell, 1986) and even strain differences in mice (Butler et al., 1988; Dwivedi et al., 1989). Certain speciessuch as guinea pigs, marmosets, and rhesus monkeys have displayed little re509
sponseor even an absenceof the induction of hepatic peroxisomes (Rodricks and Tunnbull, 1987; Lake et al., 1989). In particular, induction of peroxisomes in humans is equivocal (Hanefeld et al., 1983; Gariot et al., 1987). The increase in the number of peroxisomes and the induction of associatedenzyme activities (e.g., catalaseand oxidases)have alsobeen demonstrated in vitro in primary cultures of rodent hepatocytes (Gray et al., 1983; Reddy and Rao, 1987; Butler et al., 1988; Furukawa et al., 1988), but never in cultured human hepatocytes (Mitchell et al., 1985; Elcombe and Mitchell, 1986). 0041-008X/90
$3.00
Copylight 0 1990 by Academic Press. Inc. ALI rights of reproduction in any form reserved.
510
BICHET
In the preceding papers, a benzofuran derivative, benzbromarone, was found to produce hepatic peroxisome proliferation in rats in viva (Butler et al., 1990). Therefore, the present study was undertaken to determine whether benzbromarone would do the same in cultured rat and human hepatocytes. We report here that rat hepatocytes in primary culture exposed to benzbromarone or the reference compounds, clofibric acid and mono(2ethelhexyl)phthalate (MEHP), displayed peroxisome proliferation whereas human hepatocytes, although metabolically competent, were refractory to peroxisome proliferation. MATERIALS
AND
METHODS
.Materials Clofibric acid, insulin. hydrocortisone, palmitoyl-CoA, amino-2-ethyl1,3-propanediol, and 3,3’-diaminobenzidine (DAB) were purchased from Sigma Chemical Co. (St. Louis, MO). MEHP was supplied by Elf Aquitaine ATOCHEM Division (Paris, France). The benzofuran derivative, benzbromarone, was obtained from Sanofi Recherche (Montpellier. France). Potassium cyanide (KCN) and hydrogen peroxide (H,O,) were obtained from Prolabo (Paris, France). 3-Amino1,2,4-triazole was obtained from Fluka (Switzerland). Leibowitz L15 culture medium and glutamine were supplied by GIBCO/BRL (Paris, France); streptomycin, penicillin, and collagenase by Boehringer SA (Manheim, France). Tryptose phosphate broth was obtained from FIow Laboratories SA (France), and the fetal calf serum was supplied by IBF (France). [li4C]palmitoyl-CoA (56 mCi/mmol) was obtained from Amersham. All other chemicals were of the highest available purity and were supplied by Sigma Chemical Co., Boehringer SA, or Merck (Darmstadt, FRG).
Hepatocyte
Isolation
and Culture
Rat hepatocytes were isolated from male SpragueDawley rats by a two-step in situ perfusion technique using collagenase as described by Williams et a/. (1982). The isolated cells were washed twice by centrifugation at 5Og and resuspended in Williams’ medium E supplemented with 10% newborn calf serum + streptomycin and penicillin (WMES). Cell viability, determined by the trypan blue exclusion test, ranged between 90 and 95%. Human hepatocytes were prepared from resected liver lobe from three patients with secondary liver cancer, two of them with colon adenocarcinoma, and the third with
ET
AL.
rectum adenocarcinoma. Lobes were perfused via a branch of the portal vein with a Hepes buffer containing collagenase, according to the method developed by Fabre et a/. (1988) on whole human livers and subsequently adapted for human liver lobes (Cano et al., 1989). The viability of hepatocytes. assessed by the trypan blue exclusion test and by a light microscopic morphological analysis, ranged between 80 and 95%. The functional viability of human hepatocyte cultures from each hepatocyte preparation was assessed by measurement of the inducibility of cytochrome P450 IA-mediated 7-ethoxyresorufin-O-deethylase activity after a 72hr exposure to /3-naphthoflavone (50 @M) and 3-methylcholanthrene (50 FM), and by the increase of the specific cytochrome P450 IIIA isozyme “nifedipine oxidase” activity by rifampicin (50 FM) and dexamethazone (50 PM) (Cano et al., 1989).
Treatment eration
of Hepatocyte
Cultures
for Peroxisome
Pro&f:
Rat and/or human cellular suspensions were immediately seeded at 2.5 X lo6 viable cells per 3 ml culture medium (WMES) in 60-mm petri dishes coated with collagen and maintained at 37°C in a humidified atmosphere (5% CO2 and 85% air). Three hours after seeding for rat preparations, and 7 to 16 hr after seeding for human preparations, the culture medium and unattached cells were aspirated and replaced by fresh L15 Leibowitz medium supplemented with 10% fetal calf serum, 10e6 M insulin, 10m5 M hydrocortisone, tryptose broth phosphate (8.3%) according to Mitchell et a/. (1984). Further medium changes were performed 24, 48, and 72 hr after seeding. For each treatment, four culture dishes were used, two for enzymatic activity assay and two for morphological analysis. The test compounds, clofibric acid and MEHP, to be added to the monolayers were dissolved in dimethylfonnamide (DMF); benzbromarone was dissolved in dimethyl sulfoxide (DMSO). The concentrations used were 0.1,0.5, 0.75, I, and 2 mM for clofibric acid and MEHP. Benzbromarone was tested at 2.37, 11.85, 23.70,47.40. 59.20, and 7 1.10 @M. Test compounds were added at every 24hr medium change, unless indicated otherwise. Addition of DMF or DMSO never exceeded 10 ~1 per petri dish (this concentration (j (J. M. Lowenstein, Ed.), Vol. 72, Part D, pp. 3 15-3 17. Academic Press, San Diego. LAZAROW, P. B. (1987). Rat liver peroxisomes catalyse the P-oxidation of fatty acids. J. Biol. Chern. 253, 15221528. LOWRY, 0. H., ROSEBROUCH,N. J., FARR, A. L., AND RANDALL. R. J. (195 1). Protein measurement with the Folin-phenol agent. J Bioi. Chem. 193, 265-275. MARSMAN, D. S.. CA~LEY, R. C., CONWAY, J. G., AND POPP, J. A. (1988). Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferators di(2ethylhexyl)phthalate and 4-chloro-6-(2,3-xylidino)-2pyrimidinylthio-acetic acid (WY-14,643) in rats. Cancer Res. 48,6739-6744. MITCHELL, A. M., BRIDGES,J. W., AND ELCOMBE, C. R. (1984). Factors influencing peroxisome proliferation in cultured rat hepatocytes. Arch. Toxicol. 55, 239-246. MITCHELL, A. M., JOSEPH, K., AND ELCOMBE. C. R. (1985). Species differences in hepatic peroxisome proliferation. Hum. Toxicol. 4, 552. NOVIKOFF, A. B., AND GOLDFISCHER,S. (1969). Visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine. J. Histochem. Cytochem. 17, 675-680.
NOVIKOFF, A. B., NOVIKOFF, P. M., DAVIS, C., AND QUINTANA, N. (1972). Studies on microperoxisomes. II. A cytochemical method for light and electron microscopy. J. Histochem. Cytochem. 20, 1006-1023. NOVIKOFF, P. M., NOVIKOE, A. B., QUINTANA, N., AND DAVIS, C. (1973). Studies on microperoxisomes. III. Observations on human and rat hepatocytea J. Histo&em. Cytochem. 21, 540-558. PARZEFALL, W., SCHUPPLER,J., BARTHEL, G., MEYERROZZE, B., AND SCHULTE-HERMANN, R. (1990). Toxicological studies on a benzofuran derivative. I. A comparative study with phenobarbital on rat liver. Toxicol. Appl. Pharmacol. 106, 482-499. RAO, M. S., AND REDDY. J. K. (1989). The relevance of peroxisome proliferation and cell proliferation in peroxisome proliferator-induced hepatocarcinogenesis. Drug Metab. Rev. 21, 102-l 10.
IN RAT AND HUMAN
LIVER
517
REDDY, J. K., AZARNOFF, D. L., AND HIGNITE, C. E. ( 1980). Hypolipidemic hepatic peroxisome proliferators form a novel “class” of chemical carcinogens. Nature (London) 283,397-398. REDDY,J. K., AND LALWANI. N. D. (1983). Carcinogenesis by hepatic peroxisome proliferators: Evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. Toxicol. 12, l-58. REDDY, J. K.. LALWANI, N. D., QURESHI, S. A., REDDY, M. K., AND MOEHLE, C. M. (1984). Induction of hepatic peroxisome proliferation in non-rodent species, including primates. Amer. J. Pathol. 114, 171-183. REDDY,3. K.. AND RAO, M. S. (1987). Xenobiotic-induced peroxisome proliferation: Role of tissue specificity and species differences in response in the evaluation of the implications for human health. Arch. Toxicol. Suppl. 10,43-53.
RODRICKS.J. V., AND TUNNBULL, D. (1987). Interspecies differences in peroxisomes and peroxisome proliferation. Toxicol. Ind. Health 3, 197-212. SCHULTE-HERMANN, R., OHDE, G., SCHUPPLER.J., AND TIMMERMANN-TROSIENER, I. (198 1). Enhanced proliferation of putative preneoplastic cells in rat liver following treatment with the tumor promoters phenobarbital, hexachlorocyclohexane, steroid compounds and nafenopin. Cancer Res. 41,2556-2562. SCHULTE-HERMAW, R. (1985). Tumor-promotion in the liver. Arch. Toxicol. 57, 147-158. STERNLIEB, I.. AND QUINTANA, N. (1977). The peroxisomes ofhuman hepatocytes. Lab. Invest 36, 140-149. 140-149. STOTT. W. T. (1988). Chemically induced proliferation of peroxisomes: Implications for risk assessment. Regul. Toxicol. Pharmacol. 8, 125-l 59. WILLIAMS, G. M., LASPIA, M. F., AND DUNKEL, V. C. (1982). Reliability of the hepatocyte primary culture/ DNA repair test in testing of coded carcinogens and noncarcinogens. Mutat. Res. 97, 369-370. WILLIAMS, G. M., AND WEISBURGER, J. H. (1986). Chemical carcinogens. In Toxicology. The Basic Sciences ofPoison (C. Klaassen, M. Amdur, and J. Doull, Eds.), 3rd ed., pp. 99-173. McMillan Co., New York. WILLIAMS, G. M., MARUYAMA, H., AND TANAKA, T. (1987). Lack of rapid initiating, promoting or sequential syncarcinogenic effects of di(2-ethylhexyl)phthalate in rat liver carcinogenesis. Carcinogenesis 8, 875-880.
