The effects of genetic variation in N-acetyltransferases on 4-aminobiphenyl genotoxicity in mouse liver

Inbred, congenic and transgenic strains of mice were characterized for acetylation of p-aminobenzoic (PABA) and the carcinogen 4-aminobiphenyl (4ABP). C57Bl/6 mice have the NAT2*8 allele, A/J mice have NAT2*9 and congenic B6.A mice have NAT2*9 on the C57Bl/6 background. The first transgenic strain with human NAT1, the functional equivalent of murine NAT2, was also tested. The murine NAT2*9 allele correlated with a slow phenotype measured with the murine NAT2 selective substrate PABA. The two strains having this allele also had a lower capacity to acetylate 4ABP. A line with five copies of the human NAT1 transgene was bred for at least five generations with either C57Bl/6 or A/J mice. There was no significant change in PABA NAT activity on the C57Bl/6 background but a 2.5-fold increase was seen in hNAT1:A/J compared with A/J. The effect of variation in NATs on 4ABP genotoxicity was assessed in these strains. Twenty-four hours after exposure to a single oral dose of 120 mg 4ABP/kg, hepatic 4ABP-DNA adducts were detected by immunofluoresence in all strains. Nuclear fluorescence intensities (mean+/-S.D.) were 41.1+/-3.6 for C57Bl/6, 37.9+/-1.11 for A/J and 36.3+/-2.44 for B6.A. There was no correlation between murine NAT2 alleles and 4ABP-DNA adduct levels. Similar results were seen with the transgenic strains. The data indicate that the range of variation present in these strains of mice was insufficient to alter susceptibility to 4ABP genotoxicity. The impact of these relatively modest differences in the acetylation of the activation of 4ABP may be masked by other competing biotransformation reactions since 4ABP is a substrate for both NAT1 and NAT2. Mouse models with variation in both isoforms are needed to adequately assess the role of variation in NATs in susceptibility to 4ABP genotoxicity.     

Comparative xenobiotic metabolism capacities and pesticide sensitivity in adults of Solea solea and Solea senegalensis

The measurement of enzymatic activities involved in xenobiotic biotransformation was carried out in adults of Solea solea and Solea senegalensis. The hepatic enzymes analysed were cytochrome P450 (CYP) related activities using eight fluorometric substrates and carboxylesterases (CbE). The conjugating activities of glutathione S-transferase (GST) and UPD-glucuronosyltransferase (UDPGT) were also assessed. Specific mammalian inhibitors were used as diagnostic tools for related activities of CYP1A (α-naphthoflavone; αNF), CYP2B6 and CYP2C19 (ticlopidine) and CYP3A4 (ketoconazole). The in vitro sensitivity to organophosphorous pesticides (OP) was tested in the S10 homogenate of brain (acetylcholinesterase-AChE) and liver (CbE). Furthermore, the pesticide chlorpyrifos oxon (CLPO) was used to explore the OP sensitivity of CbE of both species in two subcellular fractions (microsomes and cytosol), using two substrates. Overall, only two parameters confirmed species differences: EROD and cytosolic CbE being significantly elevated (p < 0.05) in the common sole, S. solea. A high inhibition of CYP1A related activities using several fluorometric substrates (ER, MR and CEC) after in vitro incubation with αNF confirmed all measure CYP1A1-related activities whereas ketoconazole was more specific for BFCOD (CYP3A4). Pesticide sensitivity was similar for brain AChE but hepatic CbE had a protective role that was species and pesticide dependent.     

Genotoxic activation of 2-phenylbenzotriazole-type compounds by human cytochrome P4501A1 and N-acetyltransferase expressed in Salmonella typhimurium umu strains

Four 2-phenylbenzotriazole (PBTA)-type compounds (PBTA-4, PBTA-6, PBTA-7, and PBTA-8) were identified as major mutagens in blue cotton/rayon-adsorbed substances collected at sites below textile dyeing factories or municipal water treatment plants treating domestic waste and effluents from textile dyeing factories in several rivers in Japan. The main purpose of this study is to understand the basis of the roles of human cytochrome P450 (CYP) and N-acetyltransferases (NATs) in genotoxic activation of PBTA derivatives. We compared the induction of umuC gene expression as a measure of genotoxicity using Salmonella typhimurium TA1535/pSK1002 (parental strain), NM2009 (bacterial O-acetyltransferase-overexpressing strain) established in our laboratories. PBTA-4, PBTA-6, PBTA-7, and PBTA-8 induced the umuC gene expression more strongly in the bacterial O-acetyltransferase-overproducing strain than in the parental strain in the presence of rat S9 mix. We determined the activation of PBTA derivatives by cDNA-based recombinant (Trichoplusia ni) systems expressing human or rat cytochrome P450 enzymes (P450 or CYP) and NADPH-P450 reductase using S. typhimurium NM2009. The results showed that human recombinant CYP1A1 enzyme was much more active than CYP1A2 and CYP3A4 in the genotoxic activation of PBTA-4, PBTA-6, PBTA-7, and PBTA-8. Similarly, rat recombinant CYP1A1 enzyme catalyzed the activation of these chemicals at high rates. α-Naphthoflavone, a known inhibitor of CYP1A1, was found to inhibit genotoxic activation caused by PBTA derivatives. We further determined the activation of PBTA derivatives using S. typhimurium NM6001 (human NAT1-expressing strain), S. typhimurium NM6002 (human NAT2-expressing strain), and S. typhimurium NM6000 (O-AT-deficient parent strain) in the presence of S9 mix. PBTA-4 showed almost similar sensitivity in the NAT1-expressing strain and the NAT2-expressing strain, although NAT2-expressing strain exhibited relatively higher sensitivity to PBTA-6, PBTA-7, and PBTA-8 than NAT1-expressing strain. The results support the view that O-acetylation by human NAT1 and NAT2 enzymes is involved in the genotoxic activation of PBTA compounds. These results demonstrate for the first time that human P4501A1 and NATs (NAT1 and NAT2) contribute significantly to the activation of PBTA-type compounds to genotoxic metabolites that induce umuC gene expression in S. typhimurium tester strains.     

The influence of cytochrome P450 enzyme activity on the composition and quantity of volatile organics in expired breath

Abstract

We have previously described a method to capture, identify and quantify volatile components in expired breath. The purpose of this research is to provide a non-invasive means to measure biomarkers of metabolism in vivo. In the present studies, the effect of 1-aminobenzotriazole (ABT), an inhibitor of diverse cytochrome P450 (P450) enzymes, on the composition of volatile organic chemicals (VOCs) expired in the breath of male F-344 rats was determined in parallel with the catalytic activities and total content of hepatic P450. lntraperitoneal administration of ABT (100 mg kg-¹) to rats resulted in markedly diminished hepatic microsomal P450 content and activities. The extent of inhibition was near maximal at 4 h, at which time approximately 50% of the total P450 content, about 65% of the CYPlA2 activity, 55% of the CYP2E1 activity, and about 80% of CYP2B activity were lost. Inhibition was maintained to 48 h post-dosing, but P450 content and activities had largely been restored by day 7. Concomitant with the inhibition of P450 were corresponding increases (up to several hundred-fold) in the molar amount of volatiles appearing in the breath of ABT-treated animals, and the rebound of P450 levels was attended by corresponding decreases in the appearance of breath volatiles. These studies indicate that P450 plays a major role in the metabolism of VOCs appearing in breath, and that these chemicals can serve as markers on P450 activity in vivo.     

Induction of cytochrome P450-dependent monooxygenase in mouse liver and kidney by rutaecarpine, an alkaloid of the herbal drug Evodia rutaecarpa.

Rutaecarpine is one of the main alkaloids of an herbal remedy, Evodia rutaecarpa, which has been used for the treatment of gastrointestinal disorder and headache. Effects of rutaecarpine on hepatic and renal cytochrome P450 (CYP)-dependent monooxygenase were studied in C57BL/6J mice. Treatment of mice with rutaecarpine by gastrogavage at 50 mg/kg/day for three days resulted in 57%, 41%, 6-, and 6-fold increases of hepatic microsomal benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, 7-ethoxyresorufin O-deethylation, and 7-methoxyresorufin O-demethylation activities, respectively. However, the treatment had no effects on hepatic oxidation activities toward benzphetamine, N-nitrosodimethylamine, nifedipine, and erythromycin. In the kidney, rutaecarpine-treatment resulted in 2-fold and 42% increases of microsomal benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation activities, respectively. The treatment also increased renal 7-ethoxyresorufin O-deethylation activity to a detectable level. Immunoblot analysis of microsomal proteins showed that rutaecarpine-treatment increased the protein levels of CYP1A1 and CYP1A2 in the liver, whereas hepatic level of CYP3A-immunoreacted protein was not affected by rutaecarpine. These CYPs were not detectable in the immunoblot analyses of control and rutaecarpine-treated mouse kidney microsomes. These results indicated that rutaecarpine was a CYP1A inducer and showed potent inductive effects on both CYP1A1 and CYP1A2 in the liver.     

Sex differences in the biotransformation of 2-acetylaminofluorene in cultured rat hepatocytes

Sex-related differences in susceptability to 2-acetylaminofluorene (2-AAF) hepatocarcinogenicity and in vivo biotransformation of 2-AAF have been observed. In order to determine the contribution of hepatocytes to these differences, 2-AAF biotransformation was investigated in monolayer cultures of hepatocytes freshly isolated from male and female F-344 rats.In cultured hepatocytes from both sexes, ring and N-hydroxylated, deacetylated and conjugated metabolites were formed. The half-life of 2-AAF was similar at concentrations of 5×10^–6 and 10^–5 M; however, at 10^–4 M a slower rate was observed in cultures from males. Although the total formation of aqueous metabolites was similar, the ratio of sulfate to glucuronide conjugates of 2-AAF formed by hepatocytes from male and female rats differed. Sulfate conjugates predominated in hepatocytes from male rats, whereas in females, glucuronides predominated. The demonstration of sex-dependent variations in the rate of metabolism at a high concentration of 2-AAF and in conjugation provides evidence that in vivo differences are a function, at least in part, of the biotransformation characteristics of hepatocytes.Abbreviations 2-AAF 2-acetylaminofluorene - 2AF 2-aminofluorene - WME Williams Medium E.     

Characterization of Phase I and Phase II hepatic drug metabolism activities in a panel of human liver preparations.

The role of drug metabolism in drug discovery (lead compound selection) and the traditional role of identifying the enzymes involved in biotransformation pathways (reaction phenotyping) have both relied heavily on the availability and use of a human liver bank. The assessment of drug metabolizing enzyme activity and variability in a series of individual human livers is essential when characterizing the enzymes involved in metabolic pathways (i.e. correlation analysis). In this regard, a human liver bank of 21 samples (14 males, six females, and one unknown) was characterized with respect to the activity of several important drug metabolizing enzymes. The total CYP450 content of the livers ranged from 0.06 to 0.46 nmol/mg microsomal protein. The fold variations found in specific enzyme contents were as follows: CYP1A2 (3x), CYP2A6 (21x), CYP2C9 (8x), CYP2C19 (175x), CYP2D6 (18x), CYP2E1 (5x), CYP3A4 (18x), FMO (2.5x), UDPGT (4x), NAT (7x), COMT (5x), ST (5x), TPMT (3x), and GST (2.5x). In general, the fold variation of the Phase II enzymes was lower compared with the Phase I enzymes, with the exceptions of CYP1A2, CYP2E1, and FMO. Similar data were reviewed from other established liver banks and compared with regard to the relative variability observed in drug metabolizing capacities found in this study.     

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.     

The influence of cytochrome P450 enzyme activity on the composition and quantity of volatile organics in expired breath

We have previously described a method to capture, identify and quantify volatile components in expired breath. The purpose of this research is to provide a non-invasive means to measure biomarkers of metabolism in vivo. In the present studies, the effect of 1-aminobenzotriazole (ABT), an inhibitor of diverse cytochrome P450 (P450) enzymes, on the composition of volatile organic chemicals (VOCs) expired in the breath of male F-344 rats was determined in parallel with the catalytic activities and total content of hepatic P450. lntraperitoneal administration of ABT (100 mg kg-¹) to rats resulted in markedly diminished hepatic microsomal P450 content and activities. The extent of inhibition was near maximal at 4 h, at which time approximately 50% of the total P450 content, about 65% of the CYPlA2 activity, 55% of the CYP2E1 activity, and about 80% of CYP2B activity were lost. Inhibition was maintained to 48 h post-dosing, but P450 content and activities had largely been restored by day 7. Concomitant with the inhibition of P450 were corresponding increases (up to several hundred-fold) in the molar amount of volatiles appearing in the breath of ABT-treated animals, and the rebound of P450 levels was attended by corresponding decreases in the appearance of breath volatiles. These studies indicate that P450 plays a major role in the metabolism of VOCs appearing in breath, and that these chemicals can serve as markers on P450 activity in vivo.     

Evaluation of hepatic cytochrome P4502E1 in the species-dependent bioactivation of 4-vinylcyclohexene

4-Vinyl-1-cyclohexene (VCH), is converted by multiple forms of cytochrome P450 (CYP) to two monoepoxides (4-vinyl-1-cyclohexene 1,2-epoxide [VCH-1,2-epoxide], 4-vinyl-1-cyclohexene 7,8-epoxide [VCH-7,8-epoxide]), and 4-vinyl-1-cyclohexene diepoxide (VCD). A greater degree of formation of these epoxides by female B6C3F1 mice as compared to Fischer 344 rats correlates with the ovarian toxicity observed only in the mice. Understanding which isoforms of CYP are involved in VCH bioactivation will better explain the species-dependent ovotoxicity of VCH. Present studies focus on the role of CYP2E1, as this isoform is responsible for the bioactivation of several structurally related small molecular weight compounds, including 1,3-butadiene. Hepatic microsomes prepared from either mice or rats pretreated with the CYP inducer acetone demonstrated 2-fold increases in the formation of VCH-1,2-epoxide. However, incubations with microsomes from cyp2e1-deficient mice compared to those from wild type mice revealed no differences in the rates of bioactivation of VCH to the monoepoxides. Since repeated exposure to VCH is required for VCH-induced ovotoxicity, rodents were dosed with VCH for 5 or 10 d to observe effects on the hepatic concentration of CYP2E1 and/or associated activities. VCH pretreatment failed to increase the concentration of CYP2E1 or CYP2E1 activity in either species, as measured by immunoblotting analysis and p-nitrophenol hydroxylation. Based on these data, it is concluded that CYP2E1 does not play a role in the species differences between mice and rats in the bioactivation of VCH following repeated exposure to VCH. Other isoforms, such as those in CYP2A and CYP2B subfamilies, are likely involved in VCH bioactivation.     

Strong and weak immune responses across the same major histocompatibility barrier in rats

Inbred rat strains, Fischer 344 (F-344) and Lewis (LEW), share the serologicalAg-Bl allele and react very weakly in mixed lymphocyte culture (MLC). Despite this apparent identity atAg-B, these strains differ markedly in their immune responses to anAg-B disparate third strain Marshall 520 (M-520) (Ag-B6). F-344 recipients allowed M-520 heart grafts an extended survival, whereas LEW recipients rejected them rapidly. F-344 and M-520 showed a weak response in MLC in contrast to a strong response for LEW and M-520. F-344 produced antisera in response to injection of M-520 cells that had a relatively high antibody titer but low cytotoxic activity. F-344 responded to another strain, Buffalo (BUF) (alsoAg-B6), in a similar fashion. F-344 apparently can produce a strong allogeneic response, as it was able to rapidly reject heart grafts from (LEW x Brown-Norway) F₁ donors (LBN) (Ag-B 1/3). The low response of F-344 to M-520 probably was not due to shared antigens between the two strains because M-520 heart grafts underwent rapid rejection in LEW hosts highly tolerant to F-344. To explain the contrasting response of F-344 and LEW to theAg-B6 disparity, we propose that it is controlled by an immune-response gene(s); that F-344 has a low-responding allele and LEW has a high-responding allele. The data do not reveal a location for this proposed gene. The high-responding allele appears to be dominant, as M-520 hearts were rejected rapidly by (F-344 x LEW) F₁ recipients.     

Differential effects of fat deficiency on hepatic and pulmonary drug metabolizing enzymes in rat and mouse

In the present study, we have evaluated the effect of dietary fat deprivation on the carcinogen/drug metabolizing enzymes in rats and mice. In rats, hepatic AHH, Cyt.P-450 Cyt.b5 and Cyt.c-reductase were significantly decreased due to fat deficiency, whereas in lungs, AHH and Cyt.c-reductase were decreased without any change in Cyt.P-450 level. In mice, feeding of fat-free diet did not cause any alteration in hepatic AHH and Cyt.P-450, but the levels of Cyt.b5 and Cyt.c-reductase were significantly reduced. In contrast to liver, Cyt.P-450 and Cyt.b5 were increased in lungs. Activity of UDPGT was lowered both in liver and lungs of rats whereas GST and GSH were increased in liver only. In mice, a decrease in UDPGT and appreciable increases in GST and GSH in liver were observed. However, in lungs, UDPGT activity was enhanced by feeding fat-free diet. These observations suggest that mice and rats respond differentially to the depletion of fat in the diet.     

Schisandrin B induced antioxidant response is partly mediated by cytochrome P-4502E1 catalyzed reaction in mouse liver

In order to explore the role of cytochrome P-450 (CYP) 2E1 in schisandrin B (Sch B)-induced antioxidant and heat shock responses, the effects of Sch B treatment on hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (Hsp)25/70 expression were compared between wild-type and cyp2e1 knock-out C57B/6N mice. Cyp2e1 knock-out mice exhibited a significantly smaller degree of Sch B-induced enhancement in hepatic mtGAS when compared with the wild-type counterpart. But Hsp25/70 expression induced by Sch B was not affected. Sch B-induced enhancement of mtGAS was corroborated by the increase in hepatic mitochondrial antioxidant capacity, as assessed by in vitro measurement of oxidant production, with the enhancing effect being slightly reduced in the knock-out mice. Using liver microsomes prepared from wild-type and knock-out mice as a source of CYP, Sch B was found to be a good co-substrate for the CYP-catalyzed reaction, with the rate of NADPH oxidation observable in microsomes prepared from knock-out mice being slower. The CYP-catalyzed reaction with Sch B was associated with a concomitant production of oxidant species, with the extent of oxidant production being reduced in cyp2e1 knock-out mouse microsomes. Taken together, the results indicate that CYP2E1 is partly responsible for the hepatic metabolism of Sch B that may trigger the antioxidant response in vivo.     

Catechol-O-methyl transferase and monoamine oxidase activities in brains of mice susceptible and resistant to audiogenic seizures

The activities of catechol-O-methyl transferase (COMT), monoamine oxidase (MAO), and a methanol forming enzyme were studied in whole brain homogenates and in livers obtained from DBA/2J, C57B1/6J, and F₁ hybrid mice. DBA/2J mice are extremely susceptible to audiogenic seizures, where as C57B1/6J mice are resistant to sound-induced convulsions. C57B1/6J mice were found to have significantly higher brain levels of COMT, while MAO activities were not different in animals of these genotypes. No methanol forming activity was detected in animals of either strain. No differences were found in hepatic activities of either COMT or MAO. Pyrogallol was shown to protect DBA/2J animals against audiogenic seizures.     

Distinction between octachlorostyrene and hexachlorobenzene in their potentials to induce ethoxyphenoxazone deethylase and cause porphyria in rats and mice

The potentials of octachlorostyrene (OCS) and hexachlorobenzene (HCB) to induce liver microsomal ethoxyphenoxazone deethylation (an indicator of induction of 3-methylcholanthrene and beta-naphthoflavone-like cytochrome P-450 monoxygenase activity) and cause porphyria in male C57BL/6 and C57BL/10 mice and female F344 rats were compared. Ethoxyphenoxazone deethylation was induced much more by HCB than by OCS in both of these strains of mice (although neither OCS nor HCB greatly induced deethylation in the DBA/2 strain). In rats ethoxyphenoxazone deethylase was induced 26-fold by HCB but only four-fold by OCS, whereas dealkylation of pentoxyphenoxazone (an indicator of phenobarbital-like induction) increased 43- and 36-fold, respectively. Both chemicals were poor inducers of dealkylation of pentoxyphenoxazone in mice. When fed HCB continuously but not when given OCS, C57BL/6 and C57BL/10 mice (both after pretreatment with iron) and F344 rats developed porphyria with a depression of hepatic uroporphyrinogen decarboxylase activity. The results illustrate that in these species OCS and HCB cannot be considered as equally efficient agents for inducing ethoxyphenoxazone deethylation or causing porphyria. If these effects are mediated through binding to the aromatic hydrocarbon responsiveness (Ah) receptor, HCB would appear to have a much greater affinity than OCS despite the face that neither chemical possesses a structure currently considered to be necessary for efficient binding.     

Uroporphyria and hepatic carcinogenesis induced by polychlorinated biphenyls-iron interaction: absence in the Cyp1a2(-/-) knockout mouse

Aryl hydrocarbon receptor ligands, such as polychlorinated biphenyls (PCBs), cause inhibition of the heme biosynthesis enzyme, uroporphyrinogen decarboxylase; this leads to uroporphyria and hepatic tumors, which are markedly enhanced by iron overload in C57BL/10 and C57BL/6 strains of mice. Cyp1a2(-/-) knockout mice were used to compare the effects of CYP1A2 expression on uroporphyria and liver carcinogenesis. PCBs in the diet (100ppm) of Cyp1a2(+/+) wild-type mice caused hepatic uroporphyria, which was strongly increased by iron-dextran (800mg Fe/kg). In contrast, uroporphyria was not detected in Cyp1a2(-/-) knockout mice, although expression of CYP1A1 and CYP2B10 was greatly induced. After 57 weeks on this diet, hepatic preneoplastic foci and tumors were seen in the Cyp1a2(+/+) mice; numbers and severity were enhanced by iron. No foci or tumors were detected in Cyp1a2(-/-) mice, although evidence for other forms of liver injury was observed. Our findings suggest a link not only between CYP1A2, iron metabolism, and the induction of uroporphyria by PCBs, but also with subsequent hepatocarcinogenesis.     

Endotoxemia and hepatic injury in a rodent model of hindlimb unloading.

Hindlimb unloading (HU) is known to induce physiological alterations in various organ systems that mimic some responses observed after exposure to microgravity. In the present study, the effects of up to 4 wk of HU on the liver were assessed in male Wistar rats and two mouse strains: endotoxin-sensitive C57BL/6 mice and endotoxin-resistant C3H/HEJ mice. Plasma levels of endotoxin, a known stimulator of hepatic injury, were measured in portal and systemic blood samples. Endotoxin was elevated by approximately 50% in portal blood samples of mice and rats but was not detectable in systemic blood. This low-grade portal endotoxemia was associated with hepatic injury in rats and C57BL/6 mice as indicated by inflammation and elevated serum transaminase activities. Blood levels of the cytokine TNF-alpha were increased by approximately 50% in C57BL/6 mice; no significant elevation of this cytokine was detected in rats. Messenger RNA levels of the acute-phase proteins serum amyloid A, haptoglobin, and lipopolysaccharide binding protein were significantly enhanced after 3 wk of HU in endotoxin-sensitive rodents. In contrast, no histological changes or significant increases in serum enzyme activity were detected after HU in C3H/HEJ mice despite portal endotoxin levels of 222 +/- 83.4 pg/ml. At the 3-wk time point, expression of acute-phase proteins was not elevated in C3H/HEJ mice; however, expression after 4 wk of HU was similar to endotoxin-sensitive rodents. In conclusion, these findings indicate that HU induced mild portal endotoxemia, which contributed to the observed hepatic injury in endotoxin-sensitive rodents.     

Lack of mechanism-based inactivation of rat hepatic microsomal cytochromes P450 by doxorubicin.

Administration of the antineoplastic doxorubicin to rodents causes depression of hepatic cytochrome P450 (CYP) dependent biotransformation, an effect that has been partially attributed to the ability of doxorubicin to stimulate microsomal lipid peroxidation. Since doxorubicin can be bioactivated by the CYP/NADPH-CYP reductase system to products that bind covalently to microsomal protein, we hypothesized that doxorubicin functions as a mechanism-based inactivator of hepatic microsomal CYPs and (or) NADPH-CYP reductase under conditions in which doxorubicin-stimulated NADPH-dependent lipid peroxidation is minimized. In vitro studies were conducted with hepatic microsomes isolated from untreated and phenobarbital-treated male rats. Unlike the positive control carbon tetrachloride, doxorubicin (10 microM) did not stimulate NADPH-dependent lipid peroxidation in microsomal incubations containing EDTA (1.5 mM). Doxorubicin did not cause NADPH-dependent loss of microsomal CYP, heme, or steroid hydroxylation activities selective for CYP2A, CYP2B, CYP2C11, and CYP3A. The positive control 1-aminobenzotriazole caused marked NADPH-dependent decreases in all of these parameters. Neither doxorubicin nor 1-aminobenzotriazole caused NADPH-dependent loss of NADPH-CYP reductase activity, and neither compound altered the immunoreactive protein levels of CYP2B, CYP2C11, CYP3A, and NADPH-CYP reductase. These results indicate that a pharmacologically relevant concentration of doxorubicin does not cause direct mechanism-based inactivation of hepatic microsomal CYPs or NADPH-CYP reductase, suggesting that the ability of doxorubicin to depress hepatic CYP-mediated biotransformation in vivo is due to lipid peroxidation mediated heme destruction, altered heme metabolism, and (or) decreased expression of selected CYP enzymes.     

Paradoxical effect of sudan III on the in vivo and in vitro genotoxicity elicited by 7,12-dimethylbenz(a)anthracene

Effect of the induction of drug metabolizing enzymes by Sudan III on the in vivo and in vitro genotoxicity elicited by 7,12-dimethyl-benz(a)anthracene (DMBA) was investigated. A significant suppression of DMBA-induced micronucleated reticulocytes was observed in C57BL/6 mice treated with Sudan III intraperitoneally for 3 or 5 days before injection of the DMBA. However, the preincubation of DMBA with hepatic microsomes from Sudan III-treated rats caused a marked increase in the in vitro mutagenicity in the Ames assay, paradoxically. Sudan III was found to induce CYP 1A1, 7-ethoxycoumarin O-deethylase activity as well as both UDP-glucuronyl transferase and glutathione S-transferase activities. The increase of mutagenicity of DMBA observed in the Ames assay using hepatic microsomes from Sudan III-treated rats was inhibited by the addition of uridine 5′-diphosphoglucuronic add or reduced glutathione with cytosol. Mutagenic metabolites of DMBA formed by CYP1A1 appeared to be effectively detoxified by these phase II enzymes. The results of this study suggest that Sudan III-induced prevention of in vivo mutagenesis is due to the induction of both CYP 1A1 and detoxifying phase II enzymes. The induced CYP1A1 may accelerate formation of active metabolic intermediates, but phase II enzymes are also induced and detoxify these intermediates to inactive metabolites. This would reduce residence time of the carcinogen in the body and the time of exposure to active metabolites for target organs.