Tim J.B. Gray

Peroxisome proliferation in primary cultures of rat hepatocytes

Primary cultures of rat hepatocytes were exposed to a range of chemicals known to cause peroxisome proliferation in vivo. Peroxisomal palmitoyl-CoA oxidation and carnitine acetyltransferase (CAT) activity were increased within 12 to 24 hr of adding 0.5 mM clofibrate to the culture medium, reaching about 20 times control levels after 72 hr. Stimulation of CAT activity was dose related over a concentration range of 0.05 to 2 mM clofibrate and 0.02 to 0.2 mM mono-2-ethylhexylphthalate (MEHP). Higher concentrations of MEHP were cytotoxic. The stimulation of CAT activity and palmitoyl-CoA oxidation produced by clofibrate and MEHP was inhibited by cycloheximide. In further studies with clofibrate and a range of other known peroxisome proliferators (nafenopin, tiadenol, BR-931, Wy-14,643, and acetylsalicylic acid), induction of CAT and palmitoyl-CoA oxidation was observed with no increase in activity of another peroxisomal enzyme, D-amino acid oxidase. This differential effect on peroxisomal enzyme activity is typical of that seen in vivo. Furthermore, the relative potencies of the different peroxisome proliferators in vitro agreed well with what is known from studies in vivo. Mitochondrial and microsomal marker enzymes showed little change in activity. Electron microscopy of treated cultures revealed increased numbers of peroxisomes, some of which lacked the characteristic nucleoid. The results indicate that primary cultures of rat hepatocytes provide a rapid, sensitive means of identifying chemicals that cause peroxisome proliferation and a potentially valuable system for studies aimed at clarifying the toxicological significance of this phenomenon.

Comparative studies on nafenopin-induced hepatic peroxisome proliferation in the rat, Syrian hamster, guinea pig, and marmoset☆

Nafenopin was administered orally for 21 days to male Sprague-Dawley rats (0.5-50 mg/kg/day), Syrian hamsters (5-250 mg/kg/day), Dunkin-Hartley guinea pigs (50 and 250 mg/kg/day), and marmosets (Callithrix jacchus, 50 and 250 mg/kg/day). With the rat, and to a lesser extent in the hamster, nafenopin treatment produced dose-related increases in liver size and induction of peroxisomal (palmitoyl-CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidizing enzyme activities. In contrast, in the guinea pig and marmoset, there was no effect on liver size and only comparatively small changes were observed in these enzyme activities. Ultrastructural examination of liver sections from nafenopin-treated rats and hamsters revealed increased numbers of peroxisomes many of which lacked the characteristic crystalline nucleoid. While nafenopin had little effect on peroxisome numbers in either the guinea pig or marmoset, increases in microsomal cytochrome P450 content and mixed function oxidase activities were observed in these species. These results demonstrate marked species differences in nafenopin-induced hepatic peroxisome proliferation with the Syrian hamster being less responsive than the rat and the guinea pig and marmoset being only weakly responsive. As nafenopin is a known hepatocarcinogen in the rat, comparative long-term studies in poorly responsive species, such as the guinea pig and marmoset, may help clarify the role of organelle proliferation in the hepatocarcinogenicity of certain peroxisome proliferators.

The role of metabolism in 2-methoxyethanol-induced testicular toxicity.

The role of metabolism in 2-methoxyethanol (ME)-induced testicular toxicity has been investigated with Sprague-Dawley rats. Following administration of [14C]ME (250 mg/kg, ip) to a group of animals, there was evidence of testicular damage, identified as depletion of the spermatocyte population. Radioactivity detected in urine over 48 hr after treatment accounted for 55% of the dose. The major urinary metabolites were identified by HPLC and isotope dilution analysis, as methoxyacetic acid (MAA) and methoxyacetylglycine (accounting for 50 to 60% and 18 to 25%, respectively, of urinary radioactivity). Analysis of plasma revealed a rapid conversion of ME to MAA (t1/2 for disappearance of ME = 0.6 +/- 0.03 hr) and gradual clearance of radioactivity (t1/2 = 19.7 +/- 2.3 hr). Pretreatment of animals with pyrazole (400 mg/kg, ip) 1 hr prior to [14C]ME dosing gave complete protection against the testicular toxicity of ME. Radioactivity detected in the urine from the pyrazole-pretreated groups over 48 hr (18%) was significantly lower than in the ME-only group. The major radioactive peak co-chromatographed with ME (30 to 36% of the total urinary radioactivity). MAA and methoxyacetylglycine were not major metabolites. Analysis of plasma revealed almost complete inhibition of the conversion of ME to MAA (t1/2 for disappearance of ME = 42.6 +/- 5.6 hr, clearance of radioactivity t1/2 = 51.0 +/- 7.8 hr). The results demonstrate that metabolic activation is required for 2-methoxyethanol to exert toxicity to the male reproductive system.

Peroxisome proliferation in cultured rat hepatocytes produced by clofibrate and phthalate ester metabolites

Adult rat hepatocytes cultured for 48 h in the presence of 0.2 mM clofibrate, mono-(2-ethylhexyl)-phthalate (MEHP) or 2-ethylhexanol (2-EHA) contained increased numbers of peroxisomes. In keeping with the effects of these compounds in vivo, the peroxisome proliferation was associated with marked increases (up to 15-fold) in the activity of carnitine acetyltransferase. No such effects were produced by n-hexanol or two microsomal enzyme inducers, phenobarbital and 1,2-benzanthracene. These results suggest that cultured hepatocytes may provide a useful model system for studying chemically induced peroxisome proliferation.

Studies on the mechanism of Coumarin-induced toxicity in rat hepatocytes: comparison with dihydrocoumarin and other coumarin metabolites

Single doses of coumarin (125 mg/kg, ip) produced a depletion of hepatic nonprotein sulfhydryl groups (mainly reduced glutathione; GSH) in young male Sprague-Dawley rats after 2 hr and increased liver weight and produced hepatic centrilobular necrosis after 24 hr. Coumarin also produced time- and dose-dependent toxic effects in primary rat hepatocyte cultures. A marked reduction of GSH levels was also observed in vitro and this was not due either to the formation of oxidized glutathione (GSSG) or to the leakage of GSH and/or GSSG from the hepatocytes. Coumarin-induced toxicity in rat hepatocytes could be inhibited by the cytochrome P450 inhibitors ellipticine and metyrapone and potentiated by depleting hepatocyte GSH levels with diethyl maleate. In contrast to coumarin, dihydrocoumarin--which lacks the 3,4-double bond--produced little toxicity in rat hepatocytes either in vivo (127 and 254 mg/kg, ip) or in vitro. Similarly, coumarin was more toxic to rat hepatocytes than a number of known coumarin metabolites including 3- and 7-hydroxycoumarin and o-hydroxyphenylacetic acid. The results of these studies demonstrate a good in vivo/in vitro correlation for the effects of coumarin and dihydrocoumarin in rat hepatocytes. Furthermore, the data suggest that coumarin hepatoxicity in the rat is due to coumarin bioactivation by cytochrome P450-dependent enzymes to a toxic metabolite(s), which may be a coumarin 3,4-epoxide intermediate. GSH appears to protect against coumarin-induced toxicity possibly by the formation of conjugates with the toxic coumarin metabolite(s).

Comparative studies on di-(2-ethylhexyl) phthalate-induced hepatic peroxisome proliferation in the rat and hamster.

Young male Sprague-Dawley rats and Syrian hamsters were treated with 25-1000 mg/kg/day di-(2-ethylhexyl) phthalate (DEHP) orally for 14 days. Liver enlargement was observed in both species, the magnitude being greater in the rat than in the hamster. In the rat there was a marked dose-dependent induction of the peroxisomal marker cyanide-insensitive palmitoyl-CoA oxidation and also of carnitine acetyltransferase. Little effect was observed on the mitochondrial markers carnitine palmitoyltransferase and succinate dehydrogenase. Whereas in the rat, increased peroxisomal enzyme activities were observed after treatment with 100 and 250 mg/kg/day DEHP, much less effect was observed in the hamster even after 1000 mg/kg/day DEHP. Parallel morphological investigations demonstrated a greater increase in hepatic peroxisome numbers in the rat than in the hamster. 14C-labeled DEHP was found to be more rapidly hydrolyzed by rat than hamster hepatic and small intestinal mucosal cell preparations and differences were also observed in the absorption and excretion of oral doses of [14C]DEHP. Studies with mono-(2-ethylhexyl) phthalate (MEHP), a primary metabolite of DEHP, and a hypolipidemic drug clofibrate also resulted in a greater increase in hepatic peroxisomal enzymes in the rat compared to the hamster. The results demonstrate that while DEHP, MEHP, and clofibrate induced hepatic peroxisome proliferation in both species, there was a marked species difference in response. Comparative long-term studies in these species may thus help to clarify the role of peroxisome proliferation in the hepatocarcinogenicity of DEHP.

Peroxisome Induction Studies On Seven Phthalate Esters

Seven phthalate esters, representing a variety of chain lengths and degrees of branching in the alcohol moiety, were tested for their ability to produce peroxisome proliferation in the Fischer 344 rat. Di(2-ethylhexyl)adipate (DEHA) was tested using the same protocol and di(2-ethylhexyl)phthalate (DEHP) was run with each study as an internal control. Each ester was administered in the feed for a period of 21 days at levels of 2.5%, 1.2% and either 0.6% or 0.3%. DEHP and DEHA were also fed at levels of 0.1% and 0.01%. The animals were sacrificed and samples of liver were prepared for both light and electron microscopy. Serum samples were assayed for both triglyceride and cholesterol. The remaining portion of the liver was homogenized and assayed for cyanide-insensitive palmitoyl-CoA oxidation, lauric acid 11-hydroxylase and lauric acid 12-hydroxylase. The results show that there is approximately a ten-fold difference between the weakest and strongest esters in terms of their potency to induce changes in relative liver weight and in several of the biochemical parameters. In general, the longer chain esters were more potent than the shorter chain ones, and branched chain esters seemed more potent than straight. Several statistical analyses of the dataset have been performed and all render similar conehcsions. The results of one of these evaluations are presented elsewhere in this volume (Lin, 1987).

A quantitative study of stage-specific spermatocyte damage following administration of ethylene glycol monomethyl ether in the rat

A quantitative study has been carried out to characterize the stage susceptibility of the spermatocyte to ethylene glycol monomethyl ether (EGM) toxicity. EGM was administered as a single oral dose of 250 mg/kg body wt and rats were examined at time periods after dosing. The number of spermatocytes and round spermatids in tubules at each stage of spermatogenesis was counted. A sharp transition in susceptibility was observed between zygotene spermatocytes in stage XIV which showed no effect and pachytene spermatocytes in stage I which showed death or depletion of 70% of its population after 1 day. A similar transition was seen between dividing spermatocytes and step 1 spermatids, the latter being unaffected. There was a gradual reduction in susceptibility toward midpachytene such that cells in stages VII-XI showed no effect. Analysis of later time periods revealed no effect on spermatogonia or prepachytene spermatocytes but did indicate that midpachytene spermatocytes underwent delayed cell death after further progression through the cycle. In a separate sequential morphological study of early changes, the earliest signs of necrosis were seen 12 hr after dosing and were restricted to spermatocytes in stages V, XI, and XII. Cell death then progressed in a wave-like manner through stages XIII and XIV finally reaching stage I, 24 hr after dosing.

The ultrastructural effects of di-n-pentyl phthalate on the testis of the mature rat

A sequential morphological study has been carried out to examine the ultrastructural effects of di-n-pentyl phthalate (DPP) on the mature rat testis. A single oral dose of 2.2 g DPP/kg body wt was administered, and testes, perfuse-fixed 3-48 hr after dosing, were examined by transmission electron microscopy. By 3 hr, rarefaction of the basal Sertoli cell cytoplasm was seen and the basal plasma membranes separating adjacent Sertoli cells were thrown into a series of convoluted profiles with the appearance of interdigitating cell processes. The subjacent ectoplasmic specializations that normally face these membranes were disrupted, and by 12 hr the inter-Sertoli junctions showed numerous membrane discontinuities. The lateral processes of Sertoli cell cytoplasm, which separate germ cells, showed retraction and fragmentation, resulting in direct contact between adjacent germ cells or the isolation of germ cells unapposed by Sertoli cell plasma membrane. In addition, the ectoplasmic specializations associated with Sertoli-spermatid and Sertoli-pachytene spermatocyte junctions were often disrupted or absent. The mitochondria in the Sertoli cells were enlarged and, in some tubules, increased in number. The changes seen were restricted to tubules in the successive stages XI-XIV, I, and II of the spermatogenic cycle. Elongating spermatids (steps 12-15) showed cytoplasmic condensation and vacuolation by 12 hr and were necrotic by 24 hr. A small proportion of zygotene and early pachytene spermatocytes showed necrosis by 24 hr after dosing. By 48 hr, the cytoplasmic rarefaction and convoluted plasma membranes had regressed and ectroplasmic specializations had reformed along Sertoli-Sertoli junctions.

Effect of prolonged administration of clofibric acid and di-(2-ethylhexyl)phthalate on hepatic enzyme activities and lipid peroxidation in the rat

Male Sprague-Dawley rats were fed diets containing either 0.5% clofibric acid (CA) or 2% di-(2-ethylhexyl)phthalate (DEHP) for 2 years. Both compounds produced liver enlargement which was accompanied by the formation of liver nodules. Hepatic peroxisomal and microsomal fatty acid oxidising enzyme activities were induced in both large nodules and host tissue (i.e. tissue remaining after removal of large nodules) preparations from CA and DEHP treated rats. In contrast, little change in catalase activity was observed and the activities of cytosolic GSH peroxidase and GSH S-transferases were markedly reduced. Increased lipid peroxidation was observed by measurement of conjugated dienes in host tissue homogenates from CA and DEHP treated rats. Microsomal NADPH-dependent lipid peroxidation was also stimulated. Histological examination revealed extensive lipofuscin deposition in non-nodular, but not in nodular, tissue sections from treated rats. These results demonstrate that prolonged peroxisome proliferation can result in lipid peroxidation and that certain enzymes which metabolise hydrogen peroxide and organic hydroperoxides are either little affected or markedly inhibited.