Topically applied nitropyrenes are potent inducers of cutaneous and hepatic monooxygenases

The inducibility of skin and liver microsomal cytochrome P-450 dependent aryl hydrocarbon hydroxylase and other monooxygenases by a mixture of nitropyrenes was assessed and compared with the parent non-nitrated compound, pyrene. A single topical application of nitropyrenes to neonatal rats resulted in highly significant induction of aryl hydrocarbon hydroxylase, ethoxycoumarin O-de-ethylase, and ethoxyresorufin O-de-ethylase activities in skin and liver after 24 hours. Inducibility of the skin and liver enzymes was 3.9-5.7 fold and 1.8-10.3 fold respectively. On the other hand, aminopyrine N-demethylase, benzphetamine N-demethylase and epoxide hydrolase activities in the liver were unaffected by topically applied nitropyrenes. Furthermore, treatment with nitropyrenes produced a 1 nm shift to the blue region in the wavelength maximum of hepatic microsomal cytochrome P-450. Topically applied pyrene produced only marginal or no effects on cutaneous and hepatic enzyme activities. Our results suggest that nitration of pyrene, a relatively ineffective enzyme inducer, produces nitropyrenes which are potent inducers of hepatic and cutaneous monooxygenases and they resemble 3-methylcholanthrene in this inducing effect.     

Effects of varying inducer type and dose on hepatic monooxygenase activities in the mountain vole Microtus montanus

Hepatic monooxygenase activities of the mountain vole, Microtus montanus, were measured after i.p. injections of phenobarbital, B-naphthoflavone and Aroclor 1254 at doses ranging from 5 to 80 mg/kg. The results showed that mountain voles differed in their induction of hepatic monooxygenase activity relative to other rodents. The results also suggest substrate specificity in the detection of enzymatic induction and the importance of considering effects of varying inducer doses on hepatic monooxygenases.     

Regulation of aromatic oxidation of estradiol-17 beta in maternal hepatic, fetal hepatic and placental tissues: comparative effects of a series of inducing agents

The effects of nine separate inducers of cytochrome P-450-dependent monooxygenases on the hydroxylation of estradiol-17 beta (E2) were investigated in near-term pregnant rats. Isosafrole exhibited highly effective inducing properties in the maternal liver (20-fold and 5-fold increases in 4- and 2-hydroxylase activities respectively). Pregnenolone 16 alpha-carbonitrile produced approx 20- and 30-fold increases in measured respective rates of 4- and 2-hydroxylase activities in fetal hepatic tissues; isosafrole produced only 2-fold increases in the same reaction. Only minor changes or slight increases in estrogen hydroxylation rates were observed in maternal hepatic, fetal hepatic or placental tissues following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or other potent 3-methylcholanthrene (MC)-like inducing agents (beta-naphthoflavone, MC, caffeine). Phenobarbital exhibited relatively weak inducing properties and exposure of pregnant rats to ethanol from days 3-19 of gestation was without statistically significant effects on the parameters investigated. Rat placentas exhibited extremely low estrogen hydroxylase activities irrespective of pre-exposure of pregnant rats to the inducers studied. The results suggested separate regulatory controls for estrogen 2- and 4-monooxygenase activities even though relatively high correlation between the two reaction were generally observed in all three tissues.     

Nonadditive interactive effects of polychlorinated biphenyl congeners in rats: role of the 2,3,7,8-tetrachlorodibenzo-p-dioxin receptor

Administration of 3,3',4,4',5,5'-hexa-,3,3',4,4',5-penta-, and 2,3,3'4,4'5-hexa-chlorobiphenyl to immature male Wistar rats caused a thymic atrophy at high dose levels (1.25, 1.0, and 100 mumol/kg, respectively) and induced the hepatic cytochrome P-448 dependent monooxygenases (benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase) at both high and low (0.25, 0.01, and 5 mumol/kg, respectively) doses. In contrast, 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP) (300 mumol/kg) did not elicit any of these effects but elevated hepatic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cytosolic receptor protein levels (threefold) as previously reported. The effects of hepatic receptor modulation by 2,2',4,4',5,5'-HCBP (300 mumol/kg) on the enzyme induction activities of 3,3'4,4',5-penta-, 3,3'4,4',5,5'-hexa-, and 2,3,3',4,4',5-hexa-chlorobiphenyl were dose-dependent; no interactive effects were observed at high (toxic) doses of these compounds, whereas apparent synergistically increased hepatic microsomal monooxygenase induction activities were noted at the lower submaximal induction doses. It was concluded that the increased responsiveness of the rats was due to elevated hepatic 2,3,7,8-TCDD receptor levels.     

Differential modulation of hepatic cytochrome P-450 enzymes in rat and syrian hamster by 4′-trifluoromethyl-2,3,4,5-tetrachlorobiphenyl

The effects of a single injection (40 mg/kg) of 4′-trifluoromethyl-2,3,4,5-tetrachlorobiphenyl (CF3) on hepatic cytochrome P-450 monooxygenases were assessed in rat and syrian hamster. The CF3 treatment significantly increased the total amount of cytochrome P-450 in both species. In rats, CF3 treatment caused marked increases in ethoxyresorufin O-deethylase (EROD), arylhydrocarbon hydroxylase (AHH), and testosterone 7α-hydroxylase activities but significantly reduced the activities of benzphetamine N-demethylase (BzND), erythromycin N-demethylase (ErND), testosterone 6β, 16α, and 16β-hydroxylases, and formation of androstenedione. Administration of CF3 to hamsters strongly induced the activities of EROD, AHH, BzND, testosterone 15α, and 16α-hydroxylases, and androstenedione production, whereas ErND, testosterone 6β, and 7α-hydroxylases were decreased. Administration of CF3 to rats induced the CYP1A family proteins and CYP2A1, while CF3 reduced the level of CYP2B1, and, to a lesser extent, of CYP6β2. In hamsters, CF3 treatment significantly induced the CYP1A2, CYP2A1, CYP2A8, and CYP2B1 isozymes, whereas the CYP6β2 level was decreased. The ability of hepatic microsomes to activate aflatoxin B1 and benzo(a)pyrene was elevated by CF3 treatment in hamsters, while activation of aflatoxin B1 was decreased in microsomes from CF3-treated rats. These results showed differences in the CF3-induced pattern of rat and hamster cytochrome P-450 monooxygenases.     

Ontogeny of monooxygenase activities in the marmoset monkey (Callithrix jacchus).

The pre- and postnatal development of monooxygenases in the liver and adrenal gland of marmoset monkeys (Callithrix jacchus) was investigated. Cytochrome P450 was detected in the fetal adrenal gland, but aldrin epoxidase, ethoxycoumarin O-deethylase, and ethoxyresorufin O-deethylase activities were below detection limits. Although fetal hepatic cytochrome P450 was not detected, low activities of aldrin epoxidase and ethoxycoumarin O-deethylase, but no ethoxyresorufin O-deethylase, could be detected in fetal liver. These enzymes attained adult marmosets activities when the offspring were approximately 2 months of age.     

The responses of hepatic monooxygenases of guinea pig to cadmium and nickel

When Cd (3.58 mg CdCl₂·H₂O/kg, ip) was administered to male guinea pigs 72 h prior to sacrifice, the metal significantly inhibited the aniline 4-hydroxylase (AH) (16%), ethylmorphoneN-demethylase (EMND) (26%), and aminopyrineN-demethylase (AMND) (18%) activities and cytochrome P-450 (12%) and cytochrome b₅ (10%) levels. Cd did not alter the hepatic microsomal heme level. Cd, however, significantly increased the hepatic microsomalp-nitroanisoleO-demethylase (p-NAOD) (53%) activity. When Ni (59.5 mg NiCl₂·6H₂O/kg, sc) was administered to the guinea pigs 16 h prior to sacrifice, the metal significantly depressed AH (49%),p-NAOD (66%), EMND (47%), and AMND (37%) activities, and cytochrome P-450 (15%), cytochrome b₅ (24%), and microsomal heme (28%) levels. For the combined treatment, animals received the single dose of Ni 56 h after the single dose of Cd and then were killed 16 h later. In these animals, significant inhibitions were noted in AH (51%), EMND (47%), and AMND (30%) activities, and cytochrome P-450 (15%), cytochrome b₅ (26%), and microsomal heme (30%) compared to those of controls. In the case ofp-NAOD activity, the influence was in favor of Ni, i.e, the inhibition was about 61% by the combined treatment. These results reveal that:

Inhibition of two rat hepatic microsomal drug-metabolizing enzymes by a carcinogenic N-nitrosated pesticide: N-nitrosocarbaryl

N-Nitrosocarbaryl (N-methyl-1-naphthyl N-nitrosocarbamate) was intraperitoneally administered to male and female rats on four consecutive days at the following doses: 6.25 mg, 12.5 mg, 25 mg and 50 mg/kg body weight/day in olive oil solution; the controls received just the oil. In a second experiment, a daily intraperitoneal dose of 25 mg/kg of N-nitrosocarbaryl was given for 1, 2, 3 or 4 days; the animals were killed 24 h after the last treatment. The two following microsomal enzymatic activities were assayed: aniline aromatic hydroxylase and p-nitroanisole O-demethylase; the levels of cytochrome P-450, proteins and RNA were measured in the hepatic microsomal fraction. N-Nitrosocarbaryl is an inhibitor of the two investigated microsomal monooxygenases at doses of 25 and 50 mg/kg when administered on 4 consecutive days. During the daily administration, enzyme inhibition is seen in females after one day of treatment whereas cytochrome P-450 only becomes lowered after 4 days of administration. In males, no modification of this parameter is observed whereas the activities of microsomal monooxygenases are inhibited. These results suggest that N-nitrosocarbaryl could act on the active sites of the enzymes which metabolize aniline and p-nitroanisole.     

Postnatal development of hepatic oxidative, hydrolytic and conjugative drug-metabolizing enzymes in female horses

Little is known about the effects of aging on the hepatic drug metabolizing capacity of horses despite the relatively long lifespan characterizing this species. A wide array of cytochrome P450 (CYP)-dependent monooxygenases, carboxylesterases and transferases were assayed in liver microsomes from 50 female horses in an age range between less than 1 year to over 12 years. Rather unexpectedly, both the CYP content and the activity of NADPH cytochrome c reductase rose as a function of age. Accordingly, a general increasing trend was recorded in the rate of the in vitro metabolism of the substrates reported to be related to CYP2B-, CYP2E- or CYP3A, although, as detected by Western immunoblotting, only the levels of proteins recognized by anti-rat CYP3A- and CYP2B antibodies appeared to increase consistently. Also the carboxylesterases and uridindiphosphoglucuronyl-transferase (UGT) activity toward 1-naphthol displayed a similar trend, glutathione S-transferase accepting 3,4-dichloronitrobenzene as a substrate being the only enzyme activity showing an age-related decline. A positive correlation was also found between liver cadmium content and CYP amount as well as the activities of most monooxygenases (except for those related to CYP1A), carboxylesterases, and UGT. While confirming that a number of enzyme activities are less expressed in foals, our results contradict the general view that the drug metabolizing capacity drops in elder individuals. Although several other factors can influence the kinetics of foreign compounds in aged animals, data from this study may provide insight in understanding possible age-related differences in drug efficacy and the response to toxic substances in horses.     

Role of androgens in fetal and pubertal development

During fetal development, androgens exert long-term effects which are either organizational on specific organs during a critical phase of morphogenesis (e.g. sexual differentiation of external genitalia), or programming neural functions or enzyme activities expressed later in life. At all stages of development, which extends from fetal and neonatal stages to pubertal accomplishment, androgens also have activational effects that are immediate, multiple, reversible and dose dependent. Both types of actions are intricate during human development. This review will focus (1) on the intricate morphological and activational roles of androgens on sexual differentiation and pubertal development of the genital tract, external genitalia and mammary glands, and (2) on the organizational effects of androgens on four central nervous system functions: pituitary regulation of liver metabolism, gonadotropin secretions, sex dimorphic behavioral patterns, and 'sexualization of the brain'. If the molecular basis of the immediate androgenic action is known, depending of androgen receptor's availability and affinity, little is known of the way androgens exert their influence on either so various morphological processes or neuroendocrine imprinting.     

Nonadditive effects of combined in vivo hepatic microsomal enzyme inducers in the Wistar rat

Compounds that are known to increase the hepatic microsomal cytochrome P-450 dependent monooxygenases were administered to adult female rats, alone or in combination, to determine whether their effects on certain substrate oxidations were additive. 3-Methylcholanthrene (3-MC) and pregnenolone-16 alpha-carbonitrile (PCN), known to induce different forms of cytochrome P-450, when administered together increased benzo[a]pyrene oxidation to the same level as observed following 3-MC treatment alone. Phenobarbital (Pb) and PCN when administered concomitantly increased benzo[a]pyrene, amino-pyrine, and ethylmorphine metabolism to the same extent as seen following PCN administration alone. Both compounds are known to induce different forms of cytochrome P-450. Nonadditive effects were also observed with Pb and spironolactone, as well as with Pb and trans-stilbene oxide. Treatment of adult male rats with either PCN or 3-MC resulted in significantly smaller increases in benzo[a]pyrene oxidation than observed in adult female rats. These results suggest that oxidative metabolism in hepatic microsomes is not the sum of activities of a number of cytochrome P-450s, but may represent the activity of a single predominant hemeprotein. In addition, it appears that the oxidation of substrate by a particular cytochrome P-450, in intact microsomes, is greatly influenced by the presence of another form.     

Plasmodium berghei (ANKA): infection induces CYP2A5 and 2E1 while depressing other CYP isoforms in the mouse liver

It has been reported that malaria infection impairs hepatic drug clearance and causes a down-regulation of CYP-mediated monooxygenase activities in rodents and humans. In the present study, we investigated the effects of Plasmodium berghei infection on the activity of liver monooxygenases in female DBA/2 and C57BL/6 mice. In both mouse strains, P. berghei infection decreased activities mediated by CYP1A (EROD: DBA/2 65.3%, C57BL/6 44.7%) and 2B (BROD: DBA/2 64.3%, C57BL/6 49.8%) subfamily isoforms and increased activities mediated by 2A5 (COH: DBA/2 182.4%, C57BL/6 148.5%) and 2E1 (PNPH: DBA/2 177.8%, C57BL/6 128.5%) isoforms as compared to non-infected controls. Since malaria infection also produced an increase in ALT (273.1%) and AST (354.1%) activities in the blood serum, our findings are consistent with the view that CYP2A5 activity is induced by liver injury. An almost generalized depression of CYP-mediated activities has been found with numerous infections and inflammatory stimuli but an induction of CYP2A5 had been previously noted only in some viral hepatitis and trematode (liver fluke) infections.     

Cytochrome P-450 and chromosome damage by cyclophosphamide in LEC strain rats predisposed to hereditary hepatitis and liver cancer

LEC strain rats predisposed to hereditary hepatitis and liver cancer were examined for hepatic drug-metabolizing ability and the inducibility of chromosome damage by cyclophosphamide (CP) in somatic cells. Whereas the hepatic cytochrome P-450 contents and the activities of cytochrome P-450-catalyzed monooxygenases were lower in females than in males of both LEC and control LEA strains, male LEC rats exhibited significantly reduced cytochrome P-450 contents and monooxygenase activities compared with male LEA rats. When exposed to CP, a promutagen/procarcinogen requiring P-450-dependent metabolic activation, the frequencies of chromosome aberrations and sister-chromatid exchanges (SCEs) in bone marrow cells tended to be lower in females than in males of each strain and lower in LEC than in LEA rats of the same sex. In particular, the CP-induced SCEs were substantially lower in LEC rats. However, no such sex and strain differences were found in the SCE frequencies in regenerating hepatocytes of partially hepatectomized rats exposed to CP.     

Tissue specificity of induction of hamster monooxygenase activity by ethanol

The effects of ethanol on liver, kidney and intestine monooxygenases were studied using hamsters chronically fed with isocaloric control and ethanol-containing liquid diets. The inductive effects of ethanol on liver and kidney aniline hydroxylase activities began to approach plateau level after the animals were fed ethanol for two weeks. Intestinal aniline hydroxylation was refractory to ethanol induction. In control and ethanol-fed hamsters, CO-difference spectra of hepatic and extrahepatic microsomes differed in absorption maxima. Chronic alcohol consumption caused significant increases of cytochrome P-450 and cytochrome b5 contents of liver and kidney microsomes. The increases of the heme proteins were associated with the induction of aniline hydroxylase, N-nitrosodimethylamine demethylase and 7-ethoxycoumarin 0-deethylase activities. In contrast to the liver and kidney, intestinal microsomal cytochromes P-450 and b5 contents in ethanol-treated animals were lower than the controls. Ethanol pretreatment was without effect on intestinal monooxygenase activities toward the metabolism of aniline, N-nitrosodimethylamine, 7-ethoxycoumarin and benzo(a)pyrene. Gel electrophoresis of tissue microsomes from control and ethanol-treated hamsters revealed that ethanol treatment enhanced the intensity of the protein band(s) in the cytochrome P-450 molecular weight region in the liver and kidney, but not in the intestine. These results demonstrate that in hamsters the response of monooxygenase to ethanol may vary from tissue to tissue and it is difficult to make a generalization regarding the inducing property of ethanol. The differential effect on cytochrome P-450 may be an important factor in determining the interaction between ethanol and xenobiotic metabolism in animal tissues.     

Effect of inducers and inhibitors of monooxygenase on the hydroxylation of prostaglandins in the guinea pig. Evidence for several monooxygenases catalyzing omega- and omega-1-hydroxylation.

The incubation of prostaglandins (PG's) with liver microsomes from guinea pigs treated with inducers of monooxygenase (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), benzo[alpha]pyrene (benzpyrene), or a mixture of chlorinated biphenyls (Aroclor 1254)) exhibited marked elevation of 19-hydroxylation of PGE1, PGE2, PGA1, and PGA2 without affecting significantly 20-hydroxylation. However, with respect to effects on hydroxylation of a variety of xenobiotics, benzpyrene and Aroclor treatments differed markedly; whereas Aroclor treatment elevated the demethylation of ethylmorphine, benzphetamine, and p-chloro-N-methylaniline (PCMA), benzpyrene treatment had no effect on demethylation of ethylmorphine and only a marginal effect on that of PCMA. Both inducers elevated benzpyrene hydroxylation. By contrast, treatment with phenobarbital did not affect the hepatic microsomal PG's hydroxylation, although the hydroxylation of benzpyrene and the demethylation of ethylmorphine, benzphetamine, and PCMA were enhanced. Also, the hydroxylation of PG's by kidney cortex microsomes was not affected by either benzpyrene or Aroclor treatment. Inhibitors of monooxygenase were used to help delineate the type of monooxygenases induced. At low levels of alpha-naphthoflavone (ANF), benzpyrene hydroxylation in control- and Aroclor-treated guinea pigs was only little affected; by contrast, the same concentration of ANF markedly inhibited benzpyrene hydroxylation in benzpyrene-treated guinea pigs. On the other hand, metyrapone was most inhibitory in control guinea pigs. Support for the conclusion that benzpyrene induces in the guinea pig a hepatic monooxygenase with different characteristics than that found in control animals was provided by the observation that ANF (10 MICROM) inhibited PGE1 hydroxylation more pronouncedly in liver microsomes from benzpyrene-treated than from Aroclor-treated guinea pigs or controls. In addition, in benzpyrene and Aroclor-treated guinea pigs, ANF inhibited the (omega-1)-hydroxylation more pronouncedly than that of omega-hydroxylation. By contrast, metyrapone appeared to inhibit omega-hydroxylation more effectively than (omega-1)-hydroxylation. These results indicate that in the guinea pig, hydroxylation of PG's at the omega (20-) and omega-1 (19-) positions is catalyzed by different monooxygenases and that the inducers tested affect several hepatic monooxygenases with different specificities toward xenobiotics; however, with respect to PG's only the enzyme(s) involved in the 19-hydroxylation is affected.     

Species variations in the metabolism of liposoluble organochlorine compounds by hepatic microsomal monooxygenase: comparative kinetics in four vertebrate species

Liposoluble organochlorine compounds were used as substrates in a kinetic study of the hepatic microsomal monooxygenases of the male rat, feral pigeon (Columbia livia), frog (Rana pipiens) and rainbow trout (Salmo gairdnerii). Substrate concentrations were taken down to environmentally realistic levels. Lineweaver-Burk plots gave straight lines for both aldrin (HHDN) and the dieldrin analogue HCE. For both substrates activities followed the order rat greater than feral pigeon greater than trout over the entire concentration range. The metabolism of PCB isomers in microsomes from these uninduced animals was very slow. These results give evidence of cytochrome P-450 forms which can metabolize organochlorine compounds at low substrate concentrations.     

Androgenic control of hepatic mitochondrial metabolism in an apoda, Gegenophis carnosus (Beddome).

In vivo administration of testosterone significantly stimulated the activities of cytochrome oxidase, alpha-glycerophosphate dehydrogenase (alpha-GPDH), succinate dehydrogenase (SDH) and adenosine triphosphatase (Mg2+ ATPase), in mitochondria isolated from the liver of G. carnosus. Administration of dehydroepiandrosterone and androstenedione while significantly stimulated the activities of cytochrome oxidase and alpha-GPDH, did not change that of SDH and Mg2+ ATPase. Simultaneous injections of testosterone and actinomycin D or chloramphenicol prevented the testosterone-stimulated activities of all the oxidative enzymes studied. The results clearly document the important stimulatory role of androgens in the regulation of hepatic mitochondrial metabolism in G. carnosus.     

Formation of hydrogen peroxide and N-hydroxylated amines catalyzed by pulmonary flavin-containing monooxygenases in the presence of primary alkylamines

In atypical reaction, incubation of purified rabbit pulmonary flavin-containing monooxygenase with certain primary alkylamines results in the oxidation of NADPH and the formation of hydrogen peroxide. In addition, significant amounts of N-hydroxylated primary amine are also generated, as determined by colorimetric assay and GC/MS analysis of n-octylamine metabolites. Similar reactions appear to be catalyzed by the mouse pulmonary enzyme. In contrast, incubation of primary alkylamines with hepatic flavin-containing monooxygenases from rabbit, mouse, or pig does not result in NADPH oxidation or metabolism. Another effect of primary alkylamines is marked activation of the mouse pulmonary and pig hepatic flavin-containing monooxygenases with some substrates. The structural requirements for primary alkylamines to elicit NADPH oxidation by the rabbit pulmonary enzyme or to activate the mouse pulmonary and pig hepatic enzymes are identical. This indicates that different flavin-containing monooxygenases probably have a conserved alkylamine-binding site of defined specificity. In the case of the rabbit pulmonary enzyme, this binding may occur very close to or at the catalytic site resulting in some N-hydroxylation of the alkylamine.     

Effects of early androgens on sex-typed activities and interests in adolescents with congenital adrenal hyperplasia

The goal of this study was to examine the relation of early androgen exposure to sex-typed activities and interests in adolescence. Participants aged 9-19 years included 24 girls and 18 boys with congenital adrenal hyperplasia (CAH) and 16 unaffected sisters and 24 unaffected brothers who served as controls. Using standardized questionnaires, adolescents reported on their participation in sex-typed activities and interest in sex-typed occupations, and parents reported on the adolescents' activities. As hypothesized, girls with CAH showed sex-atypical preferences: increased interest in male-typical activities and careers and reduced interest in female-typical activities and careers compared to the unexposed control girls. These results extend findings of sex-atypical play in young girls with CAH and suggest that the sex-atypical activities and interests of females with CAH reflect direct effects of androgens on the developing brain rather than social responses to virilized genitalia. These results also suggest that population sex differences in activities and interests arise in part from sex differences in early androgens.     

Potentiation of thioacetamide hepatotoxicity by phenobarbital pretreatment in rats. Inducibility of FAD monooxygenase system and age effect

The ability of phenobarbital to induce the expression and activity of microsomal drug monooxygenases in the liver presents one of the most important issues in the field of chemical interactions and in the toxicity of xenobiotics. The model of rat liver injury induced by a single dose of thioacetamide (500 mg/kg intraperitoneally) was used to study the effect of phenobarbital (80 mg/kg/day intraperitoneally) for 5 days prior to thioacetamide. Serum parameters of liver injury such as aspartate aminotransferase activity, gamma-glutamyl transferase activity and the total bilirubin levels, as well as the activities of hepatic FAD and cytochrome P450 microsomal monooxygenases, were assayed in 2- and 12-month-old rats. Samples of blood and liver were obtained from controls (injected at 0 h with 0.5 ml of 0.9% NaCl) and at 12, 24, 48, 72 and 96 h of thioacetamide intoxication either to non-treated or phenobarbital pretreated rats. Potentiation of thioacetamide hepatotoxicity by phenobarbital pretreatment was demonstrated at morphological level, and by significant increases in the activities of serum aspartate aminotransferase and gamma-glutamyl transferase, and in the levels of total bilirubin. The extent of potentiation of thioacetamide-induced liver injury by phenobarbital pretreatment was similar in both age groups. Microsomal FAD monooxygenase activity, the enzyme responsible for thioacetamide biotransformation, was significantly enhanced (twofold) by phenobarbital pretreatment, and also underwent a further increase following thioacetamide, preceding the peak of necrosis. Cytochrome P450 monooxygenases were induced by phenobarbital pretreatment more than sixfold, and sharply decreased when phenobarbital was withdrawn and thioacetamide administered, showing at 48 h intoxication values close to basal. Phenobarbital pretreatment potentiated thioacetamide necrogenicity, and this potentiation was parallel to the induction of the microsomal FAD monooxygenase system, both by phenobarbital and by thioacetamide itself. The extent of thioacetamide-induced liver injury was significantly higher in 12-month-old rats, but the effect of phenobarbital pretreatment was similar in both age groups.