Uninduced Liver Research Articles

Inhibitory potency of some methoxyphenyl derivatives compounds on class mu GST activity in vitro

Methoxy phenyl derivative’s compounds i.e. curcumin, ferulic acid, and indomethacin have been reported to have inhibitory effects on rat’s liver glutathione S-transferases (GST) activity. Vaniline and ferulic acid have been known as degradation products of curcumin, and still have methoxy phenyl groups on the structures. Until the recent years, there is no information concerning the influences of methoxy phenyl derivative’s compounds i.e. vaniline, dimethylcurcumin and ethyl-p-methoxycinnamate on GST-activity. The aim of this research is to study the correlation of the inhibitory activity of class mu GST isolated from uninduced and phenobarbital-induced rat liver cytosol by curcumin, dimethylcurcumin, vaniline, indomethacin, ethyl pmethoxycinnamate, and ferulic acid. GST activity was measured spectrophotometrically on the conjugation of DCNB and GSH. The results showed that the inhibitory activity of mu class GST in uninduced or phenobarbital-induced rat liver GST were decreasing as follow : curcumin, dimethylcurcumin, vaniline, indomethacin, ethyl p-methoxycinnamate, and ferulic acid. Key words: methoxyphenyl derivative’s compounds, glutathione S-transferases activity.

Post-transcriptional regulation of hepatic NADPH-cytochrome P450 reductase by thyroid hormone: independent effects on poly(A) tail length and mRNA stability.

Thyroid hormone [triiodothyronine (T3)] positively regulates NADPH cytochrome P450 reductase (P450R) mRNA expression in rat liver, with P450R transcription initiation being a key regulated step. T3 is presently shown to have significant post-transcriptional effects on P450R expression. T3 increased the size of cytoplasmic P450R mRNA by approximately 105 nucleotides 12 h after T3 treatment, followed by a return to basal levels at 24 h. Primer extension analysis and Northern hybridization with 5'-untranslated region probes revealed no change in P450R mRNA 5' structure with T3 treatment. By contrast, RNase H analysis revealed a transient, T3-induced increase in P450R mRNA poly(A) tail, from approximately 100 to approximately 205 A. This increase in P450R polyadenylation, detectable in the nucleus 8 h after T3 treatment and in the cytoplasm at 12 h, was transient and was reversed by 16 h, when the T3-induced accumulation of cytoplasmic P450R mRNA was near maximal. Actinomycin D blocked the increase in P450R poly(A) tail and the induction of P450R mRNA, indicating a requirement for ongoing gene transcription for both T3 responses. T3 treatment destabilized P450R mRNA in rat liver in vivo, as shown by the T3-dependent 6-fold decrease in cytoplasmic P450R mRNA half-life, from a basal value of >or=16 h in uninduced liver to approximately 2.5 h, measured 24 h after T3 administration. These findings demonstrate that T3 increases nuclear polyadenylation of P450R RNA as a transient, early regulatory response and that this response is temporally dissociated from the subsequent decrease in cytoplasmic P450R mRNA stability.

Expression and characterization of the active molecular forms of choline/ethanolamine kinase-α and -β in mouse tissues, including carbon tetrachloride-induced liver

Choline/ethanolamine kinase (ChoK/EtnK) exists as at least three isoforms (α1, α2 and β) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the α (α1 plus α2) or the β isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the α isoform was highest in testis, whereas that for the β isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of α/β hetero-oligomers, with relatively small parts of activity expressed by α/α and β/β homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl4) was shown to induce ChoK activity 2–4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl4 was α, not β. The level of α mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the α isoform. Consequently, the composition of α/α homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl4-induced liver, whereas it was less than 20% in normal uninduced liver.

Expression and characterization of the active molecular forms of choline/ethanolamine kinase-alpha and -beta in mouse tissues, including carbon tetrachloride-induced liver.

Choline/ethanolamine kinase (ChoK/EtnK) exists as at least three isoforms (alpha1, alpha2 and beta) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the alpha (alpha1 plus alpha2) or the beta isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the alpha isoform was highest in testis, whereas that for the beta isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of alpha/beta hetero-oligomers, with relatively small parts of activity expressed by alpha/alpha and beta/beta homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl(4)) was shown to induce ChoK activity 2-4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl(4) was alpha, not beta. The level of alpha mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the alpha isoform. Consequently, the composition of alpha/alpha homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl(4)-induced liver, whereas it was less than 20% in normal uninduced liver.

Effect of dietary casein levels on activation of promutagens in the spiral Salmonella mutagenicity assay II. Studies with induced rat liver S9

In the previous study (Mutation Res., this issue), we showed that increased levels of dietary casein as the sole protein source for male F344 rats decreased the ability of the uninduced liver S9s to activate 2-aminoanthracene (2AN) to a mutagen in strain TA98 using the spiral Salmonella mutagenicity assay. No effects of dietary casein levels were noted for the ability of uninduced liver S9s to activate the promutagens aflatoxin B 1 (AFB) and benzo[ a]pyrene (BAP). In the present study, we have extended this study to include liver S9s induced with either Aroclor 1254, phenobarbital or 3-methylcholanthrene (3MC). S9s were derived from individual male F344 rats fed for 6 weeks on semisynthetic diets containing 8%, 12% or 22% methionine-supplemented casein as the sole source of protein (diets were made isocaloric by adjusting the corn starch content). Rats were housed in large, raised-bed cages by groups of three/diet/inducing agent. S9 activation mixtures were prepared at 5 mg of S9 protein/ml of S9 mix. Slopes from the linear portions of the mutagenicity dose-response curves were analyzed by ANOVA comparisons. Assays used to elucidate the phase I activities of microsomal preparations were cytochrome P-450 content, cytochrome- c reductase activity, flavin-containing monooxygenase activity, 7-thoxyresorufin O-deethylation (EROD) activity, N-demethylation of benzphetamine, and para-nitrophenol O-deethylation. Phase II activities were assayed by estimating glutathione (GSH) content and measuring the metabolism of 1-chloro-2,4-dinitrobenzene (CDNB) by glutathione S-transferase in cytosolic preparations. None of the phase I or phase II endpoints were significantly affected by dietary casein levels. In general, increasing levels of dietary casein resulted in increased body and liver wet weight and amount of S9 protein. Aroclor-induced S9s from rats led the 22% or 12% casein diet were most effective at activating AFB, depending on the lot of Aroclor used for induction: these divergent results were replicated with two groups of rats for each lot of Aroclor. The observed differences between Aroclor lots are assumed to arise from variation in the mix of PCB isomers. The Aroclor-induced S9s did not exhibit any casein-related effects for the activation of BAP or 2AN. For 3MC-induced S9s, the 12% casein diets produced S9s with the highest ability to activate AFB and BAP when standardized for protein content. Phenobarbital-induced S9s did not demonstrate any dietary casein-related effects on the activation of the three model promutagens. These results illustrate the complex interaction between dietary levels of casein, enzyme inducing agent and promutagen.

Interaction of cimetidine with cytochrome P450 and effect on mixed-function oxidase activities of liver microsomes.

The histamine H2-receptor antagonist drug, cimetidine (CM), was investigated to determine its effect on the metabolism of 'model' alkoxyphenoxazone substrates ethoxyresorufin (ER) and pentoxyresorufin (PR). The investigation was carried out under different conditions in rat liver microsomes from rodents pretreated with various classical cytochrome P450 inducers. CM exerted a dual effect on uninduced and PB-induced liver microsomal ethoxyresorufin-O-de-ethylase (EROD) activities; it was initially stimulatory but became inhibitory. However, when 3-MC-induced preparations were used. CM only exerted an inhibitory effect on EROD activity over the whole concentration range (0.01 microM-20 mM). Pre-incubating PB-induced and uninduced liver microsomes with NADPH before the addition of ER and CM decreased the stimulatory effect of CM and increased the inhibitory effect in the concentration range (5-20 mM). With 3-MC-induced preparations, the inhibition of EROD was only marginally potentiated. Overall conclusions for the diphasic effects of CM on biological activities were due to CM binding with differing affinities to different P450s. Subsequent decreases and increases in stimulation and inhibition, respectively, on pre-incubation with NADPH were thought to be due to an increased affinity of CM for reduced cytochrome P450.

Beta-naphthoflavone induction of a cytochrome P-450 arachidonic acid epoxygenase in chick embryo liver distinct from the aryl hydrocarbon hydroxylase and from phenobarbital-induced arachidonate epoxygenase.

Cytochrome P-450-mediated arachidonic acid metabolism in chick embryo liver microsomes was increased by both Ah receptor-dependent (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphthoflavone) and independent (phenobarbital) P-450 inducers. Arachidonic acid epoxides and monohydroxyeicosatetraenoic acids were increased 9-12-fold. omega-1-OH arachidonic acid was also significantly increased by TCDD and beta-naphthoflavone while omega-OH arachidonic acid, the main metabolite in uninduced livers, was decreased by all three agents. The P-450s catalyzing the enhanced arachidonate metabolism in beta-naphthoflavone- and phenobarbital-treated liver were investigated in reconstituted systems containing wholly or partially purified P-450s. beta-Naphthoflavone induced formation of a 55-kDa P-450 selective for arachidonate metabolism and for epoxygenation in particular. This P-450 was purified (beta NFAA). It was found to be distinct from a 54.5-kDa beta-naphthoflavone-induced P-450 catalyzing aryl hydrocarbon hydroxylase and 7-ethoxyresorufin deethylase (designated NF1). Mean turnover numbers for arachidonate epoxygenase, aryl hydrocarbon hydroxylase, and 7-ethoxyresorufin deethylase were 11.2, 0.56, and 0.04, respectively, for reconstituted beta NFAA and 0.33, 11.8, and 2.4 for NF1. beta NFAA and NF1 also differed in chromatography elution characteristics and N-terminal amino acid sequences. Both were low spin, with carbon monoxide binding peaks at 448 nm. The phenobarbital-induced arachidonate epoxygenation was catalyzed by P-450 fractions containing the main 48- and 49-kDa phenobarbital-induced P-450s; fractions in which the 49-kDa P-450 predominated were the most active. Turnover numbers for arachidonic acid epoxygenation were not correlated with those for aminopyrine demethylation or 7-ethoxycoumarin deethylation for P-450s from phenobarbital-treated livers or with aryl hydrocarbon hydroxylase, 7-ethoxyresorufin deethylase, or 7-ethoxycoumarin deethylase for P-450s from beta-naphthoflavone-treated livers. Also, different P-450s catalyzed the epoxygenation and the omega-hydroxylation of arachidonic acid in both beta-naphthoflavone- and phenobarbital-treated livers. The findings support a physiologic role for P-450-induced arachidonate metabolism and provide a basis for a possible link between TCDD's induction of P-450 and alterations of cellular homeostasis.

Activation and detoxification of 1, 8-dinitropyrene by mammalian hepatic fractions in the Salmonella mutagenicity assay

1,8-Dinitropyrene (DNP) is a potent, direct-acting mutagen to Salmonella typhimurium TA98 due to activation by bacterial metabolism. We have studied the effects of uninduced and Aroclor-induced rodent liver post-mitochondrial supernatant (S9), microsomes or cytosolic preparations on its activity. With the plate incorporation protocol, the reported S9-mediated inhibition of mutagenicity was confirmed and shown to be greater after induction when equivalent levels of protein were plated. Cytosol increased mutagenicity considerably whereas the inhibitory effect of S9 could be attributed to the microsomal fraction. The activating and detoxifying capacities of the various hepatic fractions derived from male Sprague-Dawley rats and female BALB/c mice were similar. In the absence of hepatic preparations, DNP mutagenicity was much lower in TA98/1,8DNP6 (deficient in transacetylase) than in TA98, although mutagenic activity could be still increased in the former strain by addition of uninduced cytosol. When DNP was pre-incubated in a liquid assay with S9 before addition of TA98, inhibition occurred and was time dependent. Thus the overall detoxifying effect of S9 is independent of bacterial metabolism. Furthermore, inhibition occurred under conditions where cytosol and microsomal components of S9 were kept together, indicating that the detoxifying effect of the microsomes predominates. The data therefore suggest that the microsomal-mediated detoxification by S9 may be due to the presence of an inhibitory component, enzymatic or otherwise, whose concentration is increased after Aroclor-induction. Nitro-reductase activities detected in TA98 and TA98/1,8-DNP6 were 10-fold higher than those in the mammalian fractions, although the activities in the latter increased to the bacterial enzyme levels after incubation of induced S9 or microsomes, but not cytosol, with an NADPH-generating system.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of a hepatic mitochondrial cytochrome P-450 active in aflatoxin B1 metabolism

We have previously shown that phenobarbital (PB) increases hepatic mitochondrial cytochrome P-450 (P-450) content and also the ability to metabolize hepatocarcinogen, aflatoxin B1 [Niranjan, B. G., Wilson, N. M., Jefcoate, C. R., & Avadhani, N. G. (1984) J. Biol. Chem. 259, 12495-12501]. In the present study, we have purified a mitochondrial-specific P-450 with an apparent molecular mass of 52 kdaltons (termed P-450mt3) from PB-induced rat liver using a combination of hydrophobic and ion exchange column chromatography procedures. Polyclonal antibody to P-450mt3 failed to cross-react with P-450mt1 and P-450mt2 purified from beta-naphthoflavone- (BNF) induced rat liver mitochondria. Furthermore, P-450mt3 shows an N-terminal amino acid sequence (Ala-Ile-Pro-Ala-Ala-Leu-Arg-Thr-Asp) different from those of both P-450mt1 and P-450mt2, as well as microsomal P-450b. The polyclonal antibody to P-450mt3 cross-reacted with a P-450 of comparable size purified from uninduced mitochondria. These two isoforms, however, showed difference with respect to catalytic properties and amino acid composition. In vitro reconstitution experiments show that P-450mt3 can actively metabolize diverse substrates including (dimethylamino)antipyrine, benzphetamine, and aflatoxin B1 but shows a low vitamin D3 25-hydroxylase activity. The mitochondrial P-450 from uninduced livers, on the other hand, shows relatively high [229 pmol min-1 (nmol of P-450)-1] vitamin D3 25-hydroxylase activity but a considerably lower ability for aflatoxin B1 metabolism and no detectable activity for (dimethylamino)antipyrine and benzphetamine metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the mutagenicity of aniline in association with norharman in the Salmonella/mammalian microscome assay

Synopsis The mutagenicity of aniline was investigated in association with norharman using Aroclor 1254-induced and uninduced liver S9 fractions. The non-mutagenicity of aniline in the Salmonella/mammalian microsome test was also re-examined under various experimental conditions. Aniline caused no increase in the number of revertants per plate in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98 at concentrations of up to 5000 mug/plate. S9 preparations from rat liver, hamster liver, rat spleen, or hamster spleen were all ineffective in activating aniline to a mutagen in tests using various concentrations of S9 in S9 mix. In contrast to the results with aniline alone, mixtures of aniline and norharman were mutagenic in strain TA1538 in the presence of S9 fractions from liver of rats pretreated with Aroclor 1254. The observed mutagenicity decreased as a function of the concentration of S9 in S9 mix. In the presence of uninduced liver S9 preparations, the mutagenicity of the mixture of the two compounds was slight (three-fold).

Organ specific metabolic activation of five extracts of indoor and outdoor particulate matter.

In this study liver and lung homogenates of untreated and Aroclor 1254-pretreated rats (Wistar) and mice (Swiss) were compared for their P-450 content and their capacity to activate extracts of airborne particulate matter, sampled indoors and outdoors. Results show that in addition to liver, lung homogenates of rat (Wistar) and mouse (Swiss) are also able to activate extracts of airborne particulate matter in a comparative way. Uninduced liver and lung homogenates showed only minor differences in activation capacity in the metabolism of airborne particles. In contrast to liver homogenates, Aroclor 1254 pretreatment of test animals did not give strong induction of metabolic activation capacity of lung homogenates. P-450 content was observed in all liver and lung homogenates of mouse and rat and in human lung homogenates. The results obtained in this study suggest that the respiratory tract may be an important site for in vivo bioactivation of respirable particles.

Differential expression and function of three closely related phenobarbital-inducible cytochrome P-450 isozymes in untreated rat liver

The levels of expression of cytochromes P-450b and P-450e (both inducible by phenobarbital (PB) and differing by only 14 of 491 amino acids) in liver microsomes from untreated male rats were separately quantitated by Western blotting with a polyclonal antibody raised against P-450b that is equally effective against P-450e (anti P-450 b e ). A protein with mobility identical to P-450e was detected in all microsomal samples. Microsomes from uninduced livers of individual male rats from five different strains exhibited only minor interstrain and interindividual variability in the expression of P-450e (17 ± 5 pmol P-450e/mg microsomal protein) with the exception of the Brown Norway strain (8.5 ± 0.5 pmol P-450e/mg). Expression of P-450b varied widely from undetectable levels (<2 pmol/mg) in most Sprague-Dawley rats to about 50% of P-450e levels in Fischer and Brown Norway strains. Anti P-450 b e inhibited total metabolism of 7,12-dimethylbenz[ a]anthracene (DMBA) by uninduced microsomes, to an extent dependent on rat strain (15–30%), predominantly through inhibition of formation of 12-hydroxymethyl-7-methyl BA (12HOMMBA) (65–85%), the major metabolite of purified P-450e. A specific activity for P-450e-dependent DMBA metabolism was calculated from four sets of microsomes where the P-450b content was either undetectable or very low (0.7–1.0 nmol/nmol P-450e/min −1). Comparable calculated activities were, however, obtained from other untreated rat liver microsomes where P-450b levels were significant. Polymorphism in P-450b was detected but did not affect total P-450b expression or the sensitivity of DMBA metabolism to anti P-450 b e . A fourth band of greater mobility than P-450b (apparent M r < 50,000), was also recognized by anti P-450 b e . The intensity of this band did not vary among individual rats or among the different strains and therefore did not correlate with the sensitivity of microsomal DMBA metabolism to anti P-450 b e . A monoclonal antibody (MAb) against P-450b (2-66-3) recognized P-450's b, b 2, and e on Western blots but did not react with this higher mobility band. MAb 2-66-3 and two other MAbs produced against P-450b inhibited 12-methylhydroxylation of DMBA by untreated rat liver microsomes to the same extent as anti P-450 b e . Following PB induction, P-450b was induced to about double the level of P-450e in most rat strains examined. This study demonstrates that cytochrome P-450e is selectively expressed in livers of untreated rats and plays a substantial role in the metabolism of DMBA, despite its relatively low level of expression (⩽3% of the total spectrally detectable P-450).

Dissociation of cytochrome P-450 inactivation and induction

Polycyclic aromatic hydrocarbons bind to a cytosolic receptor that translocates into the nucleus and induces the synthesis of mRNA for a subset of cytochrome P-450 isozymes. The failure to detect a “phenobarbital” receptor, however, suggests that the induction of P-450 b e , the isozymes induced by phenobarbital, is mediated by a less direct mechanism. One attractive alternative is that a receptor exists for an endogenous substance that is specifically turned over by the trace amounts of P-450 b e present in uninduced liver. Changes in the concentration of this substance caused by agents that inhibit P-450 b e would then trigger the induction response. We report here that suppressing the catalytic activities of P-450 b e for prolonged periods with 1-aminobenzotriazole, a mechanism-based irreversible inhibitor, neither induces P-450 b e nor interferes with induction of these isozymes at the level of transcription by phenobarbital. The results argue against mechanisms for the induction of P-450 b e keyed to the effective catalytic availability of these isozymes.

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine photolabels a substrate-binding site of rat hepatic cytochrome P-450 form PB-4.

Hepatic microsomes isolated from untreated male rats or from rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) were labeled with the hydrophobic, photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID incorporation into 3-MC- and PB-induced liver microsomal protein was enhanced 5- and 8-fold, respectively, relative to the incorporation of [125I]TID into uninduced liver microsomes. The major hepatic microsomal cytochrome P-450 forms inducible by PB and 3-MC, respectively designated P-450s PB-4 and BNF-B, were shown to be the principal polypeptides labeled by [125I]TID in the correspondingly induced microsomes. Trypsin cleavage of [125I]TID-labeled microsomal P-450 PB-4 yielded several radiolabeled fragments, with a single labeled peptide of Mr approximately 4000 resistant to extensive proteolytic digestion. The following experiments suggested that TID binds to the substrate-binding site of P-450 PB-4. [125I]TID incorporation into microsomal P-450 PB-4 was inhibited in a dose-dependent manner by the P-450 PB-4 substrate benzphetamine. In the absence of photoactivation, TID inhibited competitively about 80% of the cytochrome P-450-dependent 7-ethoxycoumarin O-deethylation catalyzed by PB-induced microsomes with a Ki of 10 microM; TID was a markedly less effective inhibitor of the corresponding activity catalyzed by microsomes isolated from uninduced or beta-naphthoflavone-induced livers.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of mouse liver stored vitamin A on the induction of mutations (Ames tests) and SCE of bone marrow cells by aflatoxin B1, benzo(a)pyrene, or cyclophosphamide

Male mice (C57BL/6J) at 2 weeks of age were divided into two groups and maintained on a vitamin A-deficient or vitamin A-(retinyl acetate) supplemented diet. After 8 weeks, the average liver vitamin A concentration of mice fed on vitamin A-deficient or -supplemented diet was 36 +/- 7 micrograms/g vs 287 +/- 22 micrograms/g, respectively. Uninduced liver S9 fractions were prepared from both groups of mice and used to activate (with cofactors) the precarcinogens aflatoxin B1 (AFB), cyclophosphamide (CPP), dimethylbenz(a)anthracene (DMBA), and benzo(a)pyrene (BP) in the Salmonella mutagenicity assay. S9 fraction prepared from both groups of mice failed to activate CPP to metabolites mutagenic in tester strains TA100 and TA1535 or to activate DMBA to metabolites mutagenic in TA100, but effectively activated AFB and BP to metabolites mutagenic in TA98. Comparison of activation activities of S9 prepared from liver of mice fed a high or low level of vitamin A was made with T98 treated with AFB or BP using three doses of S9 (50, 100, and 200 microliters/plate). S9 fractions from mice with a high liver vitamin A level were consistently less potent than S9 fractions from mice with a low liver vitamin A level in activating AFB to its mutagenic metabolites. This effect was not observed in BP-treated plates. Administration of AFB to groups of mice with a high liver vitamin A level induced significantly less SCE in bone marrow cells than did administration of AFB to mice with a low liver vitamin A level. This differential sensitivity was not observed when the two groups of mice were treated with either BP or CPP. The possible relationship between vitamin A levels in vivo and mutagenesis or carcinogenesis are discussed briefly.

Reproducibility of microbial mutagenicity assays: II. Testing of carcinogens and noncarcinogens in Salmonella typhimurium and Escherichia coli.

A total of 63 chemicals were tested for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, and Escherichia coli WP2 uvrA in a four-laboratory study. Sixty of the chemicals had been tested for carcinogenicity by the National Cancer Institute or the National Toxicology Program. All chemicals were tested for mutagenicity without metabolic activation and with liver S-9 preparations from uninduced and Aroclor 1254-induced F344 rats, B6C3F1 mice, and Syrian hamsters. The intra- and interlaboratory reproducibility of the Salmonella assay with regard to the overall judgment of mutagenic or nonmutagenic was good. The results in the E coli strain, however, exhibited a high degree of variability between laboratories. With one or two exceptions, the mutagens were detected with S-9 preparations from all three species. The uninduced liver S-9 preparations did not activate any chemicals to mutagens that were not also activated by induced S-9, but some chemicals were detected as mutagens only when induced S-9 was used. A positive mutagenic response in Salmonella was predictive of carcinogenicity 69% of the time; when equivocal carcinogens and borderline mutagens were included, the predictivity increased to 83%. Conversely, 76% of the carcinogens were mutagens. When the equivocal carcinogens were included, the proportion dropped to 75%. Relatively few chemicals (18%) were mutagenic in E coli. Not all the carcinogens induced tumors in both rats and mice, and the species-specific carcinogenicity could not be predicted from the S-9-specific mutagenicity.

Isolation of human hepatic microsomes and their inhibition by cimetidine and ranitidine.

Human hepatic microsomes were isolated from wedge biopsies of the liver from 13 patients undergoing abdominal surgery. Ultrasonic homogenisation was used to increase the yield of microsomal monooxygenase activity (7-ethoxycoumarin O-deethylase, NADPH-cytochrome c reductase), resulting in a 30% higher total enzyme activity per g liver than preparation by other techniques. In 4 individual microsomal preparations the influence of cimetidine and ranitidine on Michaelis-Menten kinetics of O-deethylation and of reductase activity were studied. Without the H2-receptor blocking drugs, enzyme kinetics of O-deethylation with a Km of 51.0 +/- 16.4 microM (n = 3) were obtained using Lineweaver-Burke plots. Both, cimetidine and ranitidine inhibited the O-deethylation; cimetidine had a five-fold higher inhibitory affinity (Ki 1.01 and 3.94 mM) to the monooxygenase than ranitidine (Ki 4.96 and 17.70 mM) in the uninduced liver. However, in liver from a patient with induced enzyme activity (Km = 478.0 microM), the Ki of ranitidine was similar to that of cimetidine (Ki ran 3.57 versus Ki cim 2.49 mM). The reductase activity was not inhibited by ranitidine and only marginally so by cimetidine. The results suggest that in human hepatic microsomes oxidative drug metabolism is inhibited by both H2-receptor antagonists. However, the inhibitory potency of the compounds seems to depend on the individual isozyme pattern of the hepatic microsomes. Thus, while cimetidine is an relatively nonspecific enzyme inhibitor, ranitidine might more selectively inhibit induced drug metabolizing enzymes.

Mutagenicity of structurally related aromatic amines in the Salmonella/mammalian microsome test with various S-9 fractions

The related monocyclic aromatic amines 2,4-diaminoanisole (DAA), 2,4-diaminopropoxybenzene (DAPB) and 2,4-diaminobutoxybenzene (DABB) were tested for mutagenic activity in Salmonella typhimurium strain TA1538, using S-9 fractions from livers, kidneys and spleens of male Wistar rats for metabolic activation. In the presence of an uninduced liver S-9 fraction, DAA was weakly mutagenic (a two- or threefold increase), and the other compounds were negative. Uninduced S-9 preparations from kidney, spleen or a mixture of kidney, spleen and liver homogenates did not activate any of the compounds. On the other hand, S-9 fractions from rat liver induced with Aroclor 1254 or with a combination of phenobarbital and 5,6-benzoflavone activated both DAA and DAPB, DAA being by far the more mutagenic of the two. S-9 preparations from mixed liver, kidney and spleen homogenates from animals pretreated with Aroclor or with phenobarbital and 5,6-benzoflavone were less effective than the liver homogenates. Aroclor-induced S-9 fractions from kidneys slightly activated DAA, but S-9 fractions from spleen were ineffective in all cases. The mutagenicity ranking of the aromatic amines was DAA > DAPB > DABB. The latter compound caused little or no increase over the control numbers of revertant colonies. The order of effectiveness of inducers was Aroclor 1254 > phenobarbital + 5,6-benzoflavone > no inducer, and that of preparations from different organs was liver > mixture of liver, kidney and spleen > kidney > spleen.

Mouse hepatic metallothionein-I gene cleavage by micrococcal nuclease is enhanced after induction by cadmium.

Micrococcal nuclease has been shown to preferentially cleave chromatin in the region of genes actively engaged in transcription. We have used this preferential cleavage to show that the metallothionein (MT) gene in adult mouse liver, when induced to produce mRNA by injection of cadmium, becomes more susceptible to nuclease cleavage. However, the MT gene in uninduced liver, and the alphafoetal protein (AFP) gene in both induced and uninduced liver, remain relatively resistant to nuclease cleavage. The AFP gene is not normally expressed in cadmium induced or uninduced liver. Thus, susceptibility of genes to nuclease cleavage appears to rise with increasing transcription of the gene.

A new assay and cellular localization for an inducible glucuronyltransferase in the embryonic chick liver

A direct, radioisotopic assay is described for the uridine diphosphate glucuronic acid (UDPGA): p-aminophenol glucuronyltransferase. The assay uses solid phase p-aminophenol-Sephadex as the glucuronyl acceptor and UDP-[ 14C]GA as the glucuronyl donor. After incubation with the enzyme, the derivatized Sephadex beads are washed in SDS-urea or with high salt concentrations to remove all labeled material except for that covalently attached to the beads. Sonicated livers from chick embryos exposed to phenobarbital for at least 5 days transfer more than ten times the glucuronic acid to derivatized beads than do uninduced livers of the same developmental age. Glucuronyl-transferase activity can be detected on intact, living cells after 5 days of phenobarbital induction, whereas sonicate activity is detectable within 3 days of induction. Suspensions of living cells can show 25% the activity found in the same suspension after all the cells are lysed by sonication.