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The current studies investigate whether synergistic or antagonistic interactions in the upregulation of CYP1 activity occur in binary mixtures of polycyclic aromatic hydrocarbons (PAHs) involving benzo[a]pyrene and five other structurally diverse PAHs of varying carcinogenic activity. Precision-cut rat liver slices were incubated with benzo[a]pyrene alone or in combination with a range of concentrations of a second PAH, and ethoxyresorufin O-deethylase, CYP1A1 and CYP1B1 mRNA levels determined. Concurrent incubation of benzo[a]pyrene with either dibenzo[a,h]anthracene or fluoranthene in liver slices led to a synergistic interaction, at least at low concentrations, in that ethoxyresorufin O-deethylase activity was statistically higher than the added effects when the slices were incubated with the individual compounds. In contrast, benzo[b]fluoranthene and, at high doses only, dibenzo[a,l]pyrene gave rise to antagonism, whereas 1-methylphenanthrene had no effect at all concentrations studied. When CYP1A1 mRNA levels were monitored, benzo[b]fluoranthene gave rise to an antagonistic response when incubated with benzo[a]pyrene, whereas all other compounds displayed synergism, with 1-methylphenathrene being the least effective. A similar picture emerged when CYP1B1 mRNA levels were determined, though the effects were less pronounced. In conclusion, it has been demonstrated that the benzo[a]pyrene-mediated upregulation of CYP1, at the mRNA and activity levels, is synergistically and antagonistically modulated by other PAHs.

The objectives of the present study were two-fold: (a) to evaluate the role of molecular shape on the interaction of polycyclic aromatic hydrocarbons (PAHs) with the Ah receptor and CYP1A1 upregulation, and (b) to evaluate the potential of PAHs to induce epoxide hydrolase and glutathione S-transferase, two major enzymes involved in their metabolism. In order to achieve these objectives, precision-cut rat liver slices were incubated with a range of concentrations of seven 5-ring isomeric PAHs, namely benzo[c]chrysene, benzo[b]chrysene, benzo[g]chrysene, dibenzo[a,j]anthracene, dibenzo[a,c]anthracene, picene and pentacene, for 24h. All compounds, with the exception of pentacene, elevated the O-deethylation of ethoxyresorufin, an activity associated with CYP1A1; induction of this enzyme by the various PAHs correlated with their avidity for the Ah receptor. None of the PAHs studied increased epoxide hydrolase activity, monitored using benzo[a]pyrene 4,5-oxide. Of the seven PAHs, only benzo[g]chrysene elevated glutathione S-transferase activity, measured using 1-chloro-2,4-dinitrobenzene or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole as substrates. No relationship could be established between length or length/width and interaction with the Ah receptor and CYP1A1 up-regulation indicating that other structural or electronic factors are likely to be more important. Finally, 5-ring PAHs are poor inducers of the epoxide hydrolase and glutathione S-transferase enzyme systems.

Scope: The aryl hydrocarbon (Ah) receptor is a ligand-activated transcription factor that is activated by many carcinogens, and its target gene products play a major role in tumour development, so that antagonists of the Ah receptor represent potential chemopreventive agents. Methods and results: Experimental evidence is presented herein that phenethyl isothiocyanate (PEITC), a phytochemical present in cruciferous vegetables, is such an antagonist. PEITC was a very weak ligand to the Ah receptor, as assessed using the chemical-activated luciferase expression (CALUX) assay, and a poor inducer of CYP1A1 mRNA levels when incubated in precision-cut rat liver slices for 24 h. It antagonised effectively, however, the interaction of benzo[a]pyrene to the receptor, being capable of preventing its binding as well as displacing it from the receptor. Moreover, PEITC suppressed in concentration-dependent manner the benzo[a]pyrene-mediated rise in rat hepatic CYP1A1 mRNA levels in rat slices. Finally, PEITC antagonised the benzo[a]pyrene-mediated increase in the O-deethylation of ethoxyresorufin in both rat and human precision-cut liver slices. Conclusion: It is concluded that PEITC is an effective antagonist of the Ah receptor in rat and human liver, and this potential may contribute to its established chemopreventive activity.

It is believed that the chemopreventive activity of cruciferous vegetables in the lung and other tissues is exclusively the result of exposure to degradation products of glucosinolates, such as the isothiocyanates, and that the parent glucosinolates make no contribution. In the present study, evidence is presented for the first time that, in rat lung, the intact glucosinolates, glucoraphanin and glucoerucin, can modulate carcinogen-metabolising enzyme systems. The glucosinolates were isolated from cruciferous vegetables and incubated (1–25μM) with precision-cut rat lung slices for 24h. Both glucosinolates, at concentrations as low as 1μM, up-regulated the O -deethylation of ethoxyresorufin and the apoprotein levels of CYP1A1 and CYP1B1; supplementation of the incubation medium with myrosinase, the enzyme that converts glucosinolates to their corresponding isothiocyanates, abolished the rise in ethoxyresorufin O -deethylase activity. In contrast, neither glucosinolate, at the concentrations studied, influenced quinone reductase activity in the lung slices, but addition of myrosinase to the glucosinolate incubations led to a marked rise in activity. Glutathione S -transferase activity, monitored using 1-chloro-2,4-dinitrobenzene as the accepting substrate, was elevated in lung slices exposed to glucoraphanin. GSTα protein levels were increased by glucoraphanin and, to a much lesser extent, glucoerucin. It may be concluded that intact glucosinolates can modulate the activity of pulmonary carcinogen-metabolising enzyme systems, and can thus contribute to the documented chemopreventive activity of cruciferous vegetables in the lung.

The chemopreventive activity of the phytochemical sulforaphane, (-)1-isothiocyanato-4R-(methylsulfinyl)-butane, present in cruciferous vegetables in substantial amounts in the form of glucosinolate, was demonstrated in animal models of cancer using the racemate, despite the fact that humans are exposed only to the R-enantiomer through the diet. Since a principal mechanism of the chemopreventive activity of sulforaphane is modulation of the carcinogen-metabolising enzyme systems, a study was conducted in precision-cut rat liver and lung slices, and in FAO cells comparing the ability of R- and S-sulforaphane to modulate these enzyme systems. R-sulforaphane elevated hepatic glutathione S-transferase and quinone reductase whereas the S-enantiomer had no effect; moreover, the R-enantiomer was more effective in up-regulating GSTα, GSTμ and quinone reductase protein levels. In the lung, both enantiomers increased the same enzyme activities with the R-enantiomer being more potent; in addition, the R-enantiomer was more effective in up-regulating GSTα and quinone reductase protein levels. Both isomers increased glutathione levels in both tissues, with R-sulforaphane being more potent. Finally, R-sulforaphane was the more effective of the two isomers in up-regulating CYP1A1/1B1 apoprotein levels in both liver and lung, and CYP1A2 in the liver. Similarly, in FAO cells the R-enantiomer was far more effective in up-regulating quinone reductase and glutathione S-transferase activities and protein levels compared with the S-isomer. These studies demonstrate clearly the superiority of R-sulforaphane, when compared with the S-enantiomer, in stimulating detoxification enzymes, and raises the possibility that the animal studies that employed the racemate may have underestimated the chemopreventive activity of this isothiocyanate.

A principal mechanism of the chemopreventive activity of isothiocyanates is detoxification of the genotoxic metabolites of chemical carcinogens through up-regulation of enzymes such as quinone reductase and the glutathione-S-transferases. In this study we report, for the first time, the potential of the aromatic isothiocyanate, phenethyl isothiocyanate (PEITC) to modulate these enzymes in human liver from four donors, in comparison with rat liver. Precision-cut human and rat liver slices were incubated with PEITC at concentrations that can be achieved in plasma following dietary intake. Glutathione-S-transferase activity increased in rat slices whereas in human slices activity rose only in three of the four donors. At the protein level, a marked rise in GSTα was seen in one of the human donors whereas much less pronounced elevation was noted in the other three. Quinone reductase activity doubled in rat liver slices incubated with PEITC, and was accompanied by an increase in protein expression. Only in one of the human donors was activity and expression of quinone reductase elevated. These studies illustrate that there are very pronounced differences in the response of human liver to PEITC, indicating that the chemopreventive effect of isothiocyanates may not be manifested in all individuals.

The objective of this study was to evaluate the potential of the structurally related aliphatic isothiocyanates erucin and sulforaphane to modulate the pulmonary carcinogen-metabolising enzyme systems in rat lung, a target organ of their chemopreventive activity. Precision-cut rat lung slices were prepared and incubated for 24 h with a range of concentrations of either erucin or sulforaphane, up to 50 μM. Neither compound modulated the O-deethylation of ethoxyresorufin whereas they elevated markedly CYP1A1 and, to a lesser extent, CYP1B1 apoprotein levels. Neither compound influenced the O-depentylation of pentoxyresorufin or CYP2B apoprotein levels, but sulforaphane caused a modest increase in CYP3A2 apoprotein levels. Pulmonary quinone reductase activity, monitored using 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide as substrate, was markedly up-regulated by both compounds and was paralleled by a similar rise in protein levels. Both compounds increased cytosolic glutathione S-transferase activity, measured using 1-chloro-2,4-dinitrobenzene as the accepting substrate; a modest rise was seen in GSTα protein levels, determined immunologically, whereas GSTπ levels were un-affected by the same treatment. Finally, both erucin and sulforaphane increased total glutathione concentration in lung cytosol. It is concluded that these aliphatic isothiocyanates have the potential to antagonise the carcinogenicity of pulmonary carcinogens by stimulating the in situ detoxication of their DNA-binding genotoxic metabolites.

Rats were fed diets supplemented with phenethyl isothiocyanate (PEITC) at 0.06 (low dose, dietary intake level), 0.6 (medium dose) and 6.0 μmole/g (high dose), and xenobiotic-metabolising enzymes were monitored in liver, lung and kidney. At the low dose, inhibition of the hepatic O -dealkylation of ethoxy- and methoxyresorufin was noted, whereas at the high dose increases in the O -depentylation of pentoxyresorufin and O -debenzylation of benzyloxyquinoline were observed, whereas p -nitrophenol hydroxylase was inhibited. Hepatic bioactivation of 2-amino-3-methylimidazo-[4,5- f ]quinoline to mutagens was not influenced by the PEITC-treatment. In the lung, at the high dose, ethoxyresorufin dealkylation was elevated and that of pentoxyresorufin suppressed; no significant changes were seen in the kidney. Quinone reductase was markedly elevated at all doses in liver, but the lung enzyme was refractive whereas in the kidney a modest rise was observed at the high dose. Hepatic glutathione S -transferase activity was stimulated by PEITC-treatment, but no effect was evident in the lung or kidney. It is concluded that the effects of PEITC on xenobiotic-metabolising systems are dose- and tissue-dependent, with the liver being the most sensitive and the lung generally resistant. Increased detoxication rather than cytochrome P450 inhibition is the likely mechanism of the chemopreventive activity of PEITC.

Exposure of precision-cut rat liver slices to six structurally diverse polycyclic aromatic hydrocarbons, namely benzo[a]pyrene, benzo[b]fluoranthene, dibenzo[a,h]anthracene, dibenzo[a,l]pyrene, fluoranthene and 1-methylphenanthrene, led to induction of ethoxyresorufin O-deethylase, CYP1A apoprotein and CYP1A1 mRNA levels, but to a markedly different extent. In liver slices, constitutive CYP1A1 mRNA levels were higher, as well as being markedly more inducible by PAHs, compared with CYP1B1, a similar profile to that observed in human liver slices following exposure to the PAHs. Increase in ethoxyresorufin O-deethylase and in CYP1A1 apoprotein levels was also observed when precision-cut rat lung slices were incubated with the same PAHs, the order of induction potency being similar to that observed in liver slices. Under the same conditions of exposure, CYP1B1 apoprotein levels were elevated in the lung. Up-regulation of CYP1A1 by the six PAHs correlated with their affinity for the Ah receptor, determined using the chemical-activated luciferase expression (CALUX) assay. It may be concluded that (a) precision-cut liver and lung slices may be used to assess the CYP1 induction potential of chemicals at the activity, apoprotein and mRNA levels; (b) rat is a promising surrogate animal for human in studies to evaluate CYP1 induction potential; (c) CYP1A1 is far more inducible than CYP1B1 in both rat liver and lung; (d) CYP1 up-regulation by PAHs is related to their affinity for the Ah receptor, and finally (e) computer analysis revealed that the ratio of molecular length/width is an important determinant of CYP1 induction potency among equiplanar PAHs.

The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels. Pulmonary CYP1B1 apoprotein levels and hepatic mRNA levels were similarly enhanced. Elevated epoxide hydrolase and glutathione S-transferase activities were also observed in liver slices following incubation for 24h; similarly, a rise in apoprotein levels of both enzymes was evident, peak levels occurring at the same time point. When mRNA levels were monitored, a rise in the levels of both enzymes was seen as early as 4h after incubation, but maximum levels were attained at 24 h. In lung slices, induction of epoxide hydrolase by benzo[a]pyrene was observed after a 24-h incubation, and at a concentration of 1 microM; a rise in apoprotein levels was seen at this time point. Glutathione S-transferase activity was not inducible in lung slices by benzo[a]pyrene but a modest increase was observed in hepatic slices. Collectively, these studies confirmed CYP1A induction in rat liver slices and established that CYP1B1 expression, and epoxide hydrolase and glutathione S-transferase activities are inducible in precision-cut tissue slices.

Theaflavins and theafulvins, a fraction of thearubigins, were isolated from aqueous infusions of black tea, and their effects on the hepatic and intestinal cytochrome P450 system, and on the glutathione S-transferase, epoxide hydrolase, glucuronosyl transferase and sulphotransferase enzyme systems were investigated in rats following oral intake for four weeks. Neither theafulvins nor theaflavins influenced cytochrome P450 activity in the liver as exemplified by the O-dealkylations of methoxy-, ethoxy- and pentoxyresorufin, the hydroxylations of lauric acid and p-nitrophenol, and the N-demethylation of erythromycin; similarly, hepatic xenobiotic conjugation systems were unaffected. In the intestine, both polyphenolic fractions markedly suppressed the O-deethylation of ethoxyresorufin and this was accompanied by a decrease in the CYP1A1 apoprotein levels. Probing intestinal microsomes with antibodies to CYP2E1 revealed the presence of a single band in the cytochrome P450 region whose intensity was lower in the polyphenol-treated animals. Immunoblot analysis utilising antibodies to CYP3A showed that the treatment with theafulvins and theaflavins reduced the apoprotein levels. A single band in the cytochrome P450 region was evident when the intestinal microsomes were probed with antibodies to CYP4A1 but the level of expression was not affected by the treatment with the black tea polyphenols. Finally, treatment of the rats with theaflavins had no effect on any of the intestinal conjugating enzymes studied, but treatment with theafulvins led to inhibition of glucuronosyl transferase activity.

Cytochrome P450 expression in cervine liver was investigated using chemical probes and Western blot analysis, and compared with the rat. Deer liver, when compared with rat liver, was characterised by high ethoxyresorufin O -deethylase, coumarin 7-hydroxylase and, to a lesser extent, erythromycin N -demethylase activities; in contrast, deer liver exhibited low debrisoquine 4-hydroxylase, chlorzoxazone 6-hydroxylase and, particularly, lauric acid hydroxylase activities. Ethoxyresorufin O -deethylase activity in deer was markedly inhibited by α-naphthoflavone, but was relatively resistant to inhibition by furafylline. Coumarin 7-hydroxylase was inhibited by 8-methoxypsoralen. Western blot analysis using antibodies to rat CYP1A recognised a single, highly expressed protein. Kinetic analysis indicated that a single enzyme is likely to be responsible for the high ethoxyresorufin O -deethylase activity in deer liver. Probing of cervine hepatic microsomes with antibodies to rat CYP2A2 showed that apoprotein levels were higher in the deer compared with the rat. Eadie–Hofstee plot analysis indicated that more than one enzyme catalyses the 7-hydroxylation of coumarin. Western blot analysis using antibodies to rat CYP2B, rat CYP2C11, human CYP2D6, rat CYP3A and rat CYP4A1 revealed in each case the presence of single, poorly expressed, proteins in deer liver. In contrast, when antibodies to rat CYP2E1 were used, a highly expressed single protein was observed. Cervine hepatic microsomes metabolised testosterone to generate androstenedione and a number of hydroxylated products, the major hydroxylation sites being the 2β-, 6β- and possibly the 12-position. In summary, this is the first study showing that deer liver expresses all xenobiotic-metabolising cytochrome P450 families, but the level of expression differs from that of the rat.

The results of quantitative structure-activity relationship (QSAR) studies on six series of compounds exhibiting indirect mutagenic activity are reported. These findings demonstrate the importance of frontier orbital energies and, in some cases, frontier orbital electronic populations to overall mutagenicity in diverse polyaromatic hydrocarbons, benzidines and aminobiphenyls, benzonitrofurans, nitrogenous cooked-food mutagens, benzanthracenes, and chrysenes. The correlations between structural parameters and mutagenic potency vary from R=0.81 to R=0.97, and these findings are discussed in the context of possible molecular mechanisms of mutagenicity. In particular, it is generally regarded that cytochrome P450-mediated activation of polyaromatic hydrocarbons and their amino derivatives plays an important role in mutagenic activity. In this respect, it is apparent that enzymes of the cytochrome P4501 (CYP1) family are closely associated with the metabolic activation of polyaromatic mutagens and carcinogens via the generation of reactive intermediates (usually electrophilic in nature) that attack DNA. The findings presented in this study indicate that QSAR analyses on several series of compounds are consistent with the known evidence of procarcinogen activation mechanisms, particularly for polyaromatic hydrocarbons and their heterocyclic/amino derivatives, pointing to the importance of frontier orbital energy values in particular. Teratogenesis Carcinog. Mutagen. Suppl. 1:187–193, 2003. © 2003 Wiley-Liss, Inc.

The objective of the present studies was to define the enzyme systems catalysing the 6-hydroxylation of melatonin, by monitoring the levels of 6-sulphatoxymelatonin in rat hepatic postmitochondrial preparations and in precision-cut liver slices. Melatonin 6-hydroxylase activity was localized in microsomes and was supported by NADPH, but not NADH. Treatment of rats with beta-naphthoflavone more than tripled 6-sulphatoxymelatonin formation from melatonin, but gave rise only to a moderate increase (25%) in the sulphate conjugation of 6-hydroxymelatonin. Treatment of rats with phenobarbitone, acetone, dexamethasone and clofibrate did not increase 6-sulphatoxymelatonin generation when either melatonin or 6-hydroxymelatonin served as substrates. Of a number of cytochrome P450 inhibitors investigated, only furafylline inhibited markedly the conversion of melatonin to 6-sulphatoxymelatonin without any concomitant effect on the sulphoconjugation of 6-hydroxymelatonin. When liver slices were incubated with melatonin, treatment of rats with beta-naphthoflavone, and to a lesser extent phenobarbitone, elevated the levels of 6-sulphatoxymelatonin in the culture medium. No such increase was seen when slices from beta-naphthoflavone-treated rats were incubated with 6-hydroxymelatonin, whereas a modest increase was seen with slices from phenobarbitone-treated rats. Treatment of rats with acetone, dexamethasone or clofibrate failed to modulate the levels of 6-sulphatoxymelatonin generated from either melatonin or 6-hydroxymelatonin. Molecular modelling analysis revealed that melatonin had a high area/depth(2) ratio, displayed characteristics of CYP1A2 substrates and could be readily accommodated into the human CYP1A2 active site in a position favouring 6-hydroxylation. Collectively, all the above data provide strong experimental evidence that CYP1A2 is an important catalyst of the 6-hydroxylation of melatonin.

Experimental evidence is presented for the presence in the liver of rats and mice of an Aroclor 1254-inducible, NADH-dependent enzyme system that can catalyse the bioactivation of aromatic and heterocyclic amines to genotoxic metabolites. It differs from the established microsomal cytochrome P450 and flavin monooxygenase systems in its response to treatment with cytochrome P450 inducing agents, optimum protein concentration and in vitro modulation by DMSO. The mutagenic metabolites generated by the NADH-supported system appear to be similar to those generated by the NADPH-mediated systems. Mutagenicity of the aminocompounds in the presence of either cosubstrate was less pronounced in an O-acetyltransferase-deficient bacterial strain, implying the presence of hydroxylamines. Moreover, glutathione potentiated the mutagenic response of both the NADH- and NADPH-generated metabolites. Cytochrome c suppressed markedly the NADPH-dependent mutagenicity of aromatic amines but had no such effect in the presence of NADH. Similarly, antibodies to cytochrome P450 reductase markedly inhibited the NADPH-, but not the NADH-dependent bioactivation of the aromatic amine 2-aminoanthracene. The cytochrome P450 suicide inhibitor, 1-aminobenzotriazole, decreased the mutagenicity of both, the NADH- and NADPH-mediated bioactivation of the aminocompounds. The above findings raise the possibility that a cytochrome P450-like protein, that can receive electrons from NADH, possibly through cytochrome b5 reductase, is present in the hepatic microsomes of rats and mice, and is capable of catalysing the bioactivation of aromatic amines through N-hydroxylation. Such a hypothesis is supported by the findings that NADH could support the O-dealkylation of 7-methoxy- and 7-ethoxy-resorufin, in the absence of NADPH. Finally the NADH-dependent bioactivation of aromatic amines was induced markedly by Aroclor 1254 and benzo(a)pyrene in Ah responsive, but not Ah nonresponsive, mice indicating that it is associated with the Ah locus.

Aqueous extracts of green tea, at concentrations of 2.5. 5.0 and 7.5%, were administered to rats as the sole drinking fluid for 4 weeks. Hepatic glutathione S-transferase (GST) activity, determined using 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB) as substrates, and UDP-glucuronosyl transferase activity, determined using 2-aminophenol as substrate, were induced but the effect was not always dose dependent. At the two highest doses, hepatic catalase activity was inhibited. In a second study, animals were exposed for 4 weeks to aqueous extracts (2.5%, v/v) of green tea, black tea (which has a much lower content of flavanols compared with green tea) and decaffeinated black tea. Treatment with the black tea enhanced GST activity, whether monitored using CDNB or DCNB, and the glucuronidation of 2-aminophenol. Treatment with decaffeinated black tea failed to modulate any of these activities, whereas treatment with green tea only enhanced the glucuronidation of 2-aminophenol. Finally, at this concentration of tea extract administration, black and decaffeinated black tea, but not green tea, suppressed catalase activity. It is concluded that neither flavanols nor caffeine are responsible for the induction of hepatic Phase II activities and inhibition of catalase activity in the rat.

Using 1- and 2-aminonaphthalene as model substrates, we investigated the effect of insertion of a second amino group on mutagenicity, binding to the cytosolic Ah receptor and CYP1A inducibility, and the effects were compared to those elicited by 3,3'-diaminobenzidine and 1-naphthylethylenediamine. 1,5- and 1,8-diaminonaphthalene were effective inducers of CYP1A activity, more potent than 1-aminonaphthalene. 2,3-Diaminonaphthalene was also an inducer of CYP1A, but the effect was similar to that elicited by 2-aminonaphthalene. In contrast, 3,3'-diaminobenzidine and 1-naphthylethylenediamine did not induce CYP1A activity. All aminonaphthalenes displaced [3H]TCDD from the Ah receptor, whereas 3,3'-diaminobenzidine and 1-naphthylethylenediamine failed to do so. The latter two compounds did not elicit a mutagenic response in the Ames test. Introduction of a second amino group at the 3-position of 2-aminonaphthalene did not modulate its mutagenicity. In the case of the non-mutagenic 1-aminonaphthalene, introduction of a second amino group at position 5 had no effect but when it was incorporated at position 8, mutagenic potential was conferred to the molecule. Computer modelling of the putative active site of CYP1A2 revealed that 1,5-diaminonaphthalene is orientated so that the distance of the second amino group from the iron-oxene is 4.037 A while in the case of 1,8-diaminonaphthalene the distance is shorter, 2.744 A, favouring its activation through N-hydroxylation. Of the compounds studied, 1,8-diaminonaphthalene and, to a lesser extent, 2,3-diaminonaphthalene autoinduced their activation. It is concluded that insertion of a second amino group at the 5- or 8-position of 1-aminonaphthalene may enhance biological activity but in the case of 2-aminonaphthalene insertion of a second amino group at position 3 had no major effect.

A quantitative structure-activity relationship (QSAR) in a homologous series of alkoxy methylenedioxybenzenes (MDBs) is reported. Measurements of molecular dimensions from computer-generated space-filling structures have provided values for the shape parameter area/depth2. These have been shown to correlate with the extent of inhibition of ethoxyresorufin O-deethylase activity by a series of MDBs. The implication of this is that the MDB nucleus fits the cytochrome P4501 substrate binding site and that this ability decreases with increase in the alkyl chain length of the alkoxy substituent. These findings are in agreement with previous results relating to the spatial dimensions of the cytochrome P4501 binding site, showing that substrate specificity can be rationalized in terms of overall molecular shape.

Most xenobiotic-metabolising enzyme systems, and in particular the cytochromes P450, may be markedly up-regulated following exposure to inducing agents. A rise in activity of these enzyme systems may lead to drug interactions with adverse or even fatal outcomes. Furthermore, modulation of these enzyme systems may bring about a profound change in an individual's susceptibility to a chemical toxin. The use of precision-cut tissue slices as an in vitro system allows the rapid and facile investigation of the potential of a chemical to induce cytochrome P450 and other xenobiotic-metabolising enzymes in different animal species, as well as in humans. Various tissues (e.g. liver, lung, intestine) can be used, and the effect of chemicals investigated in a concentration-dependent manner, and employing a number of endpoints. Keywords: precision-cut tissue slices; cytochrome P450; xenobiotic metabolism; enzyme induction; drug metabolism; phase II metabolism

1.Induction of the drug-metabolising enzyme systems is a major cause of clinically relevant drug interactions. The potential of a drug to engage in such interactions may be assessed in in vitro systems such as precision-cut tissue slices. 2.Precision-cut tissue slices have a number of important advantages compared with the use of cells in culture, especially when tissues with a heterogeneous cellular population are concerned. 3.Extensive studies concerned with the induction of cytochrome P450 and Phase II enzymes in precision-cut tissue slices from various animal and human tissues, such as liver, lung and intestine, using established inducing agents have revealed that slices mimic the in vivo situation. 4.Precision-cut tissue slices is a reliable, informative and cost-effective technique for assessing the potential of chemicals to up-regulate drug-metabolising enzyme systems.

As the Ah receptor target gene products play a critical role in chemical carcinogenesis, antagonists are considered as potential chemopreventive agents. It is demonstrated in this paper that the isothiocyanates R,S-sulforaphane and erucin are non-competitive antagonists of the aryl hydrocarbon (Ah) receptor. Both isothiocyanates were poor agonists for the receptor and elevated CYP1A1 mRNA levels only modestly when incubated with precision-cut rat liver slices. In contrast, the classical Ah receptor agonist benzo[a]pyrene was a potent inducer of CYP1A1 mRNA levels, with this effect being effectively antagonized by the two isothiocyanates. In further studies, it was demonstrated that R,S-sulforaphane could both prevent the interaction of and displace already bound benzo[a]pyrene from the Ah receptor, but no concentration dependency was observed with respect to the isothiocyanate. Both erucin and R,S-sulforaphane antagonized the benzo[a]pyrene-mediated increase in the CYP1A-mediated O-deethylation of ethoxyresorufin in rat precision-cut liver slices. Of the two isomers of R,S-sulforaphane, the naturally occurring R-isomer was more effective than the S-isomer in antagonizing the activation of the Ah receptor by benzo[a]pyrene. Antagonism of the Ah receptor may be a major contributor to the established chemoprevention of aliphatic isothiocyanates.

A mechanism of action of chemopreventive glucosinolates/isothiocyanates, established largely in vitro, is to modulate carcinogen-metabolizing enzymes. Extrapolation in vivo involves relating in vitro concentrations to plasma/tissue concentrations attained in vivo, thus assuming that even transient exposure modulates enzyme activity. To test this hypothesis, precision-cut rat liver slices were incubated with glucosinolates for up to 24 h, and the O-dealkylation of methoxyresorufin and ethoxyresorufin was determined; increased activities were observed only at incubations of at least 6 h. To evaluate phase II enzymes, isothiocyanates, namely, sulforaphane, erucin, and phenethyl isothiocyanate, were similarly incubated; quinone reductase increased after incubation for 6 h or longer. When glutathione S-transferase was monitored, the phenethyl isothiocyanate-manifested rise necessitated at least a 6 h incubation, whereas in the case of sulforaphane and erucin, the activity was elevated after only 2 h. It is inferred that a rise in carcinogen-metabolizing enzymes by glucosinolates/isothiocyanates necessitates tissue exposure of at least 6 h. © 2012 American Chemical Society.

The objective of this study was to establish whether the phytochemical glucoraphasatin, a glucosinolate present in cruciferous vegetables, and its corresponding isothiocyanate, 4-methylsulfanyl-3-butenyl isothiocyanate, up-regulate enzymes involved in the detoxification of carcinogens and are thus potential chemopreventive agents. Glucoraphasatin and myrosinase were isolated and purified from Daikon sprouts and Sinapis alba L., respectively. Glucoraphasatin (0-10 μM) was incubated for 24 h with precision-cut rat liver slices in the presence and absence of myrosinase, the enzyme that converts the glucosinolate to the isothiocyanate. The intact glucosinolate failed to influence the O-dealkylations of methoxy- and ethoxyresorufin or the apoprotein expression of CYP1 enzymes. Supplementation with myrosinase led to an increase in the dealkylation of methoxyresorufin, but only at the highest concentration of the glucosinolate, and CYP1A2 expression. In the absence of myrosinase, glucoraphasatin caused a marked increase in epoxide hydrolase activity at concentrations as low as 1 μM paralleled by a rise in the enzyme protein expression; at the highest concentration only, a rise was also observed in glucuronosyl transferase activity, but other phase II enzyme systems were unaffected. Addition of myrosinase to the glucoraphasatin incubation maintained the rise in epoxide hydrolase and glucuronosyl transferase activities, further elevated quinone reductase and glutathione S-transferase activities, and increased total glutathione concentrations. It is concluded that at low concentrations, glucoraphasatin, either intact and/or through the formation of 4-methylsulfanyl-3-butenyl isothiocyanate, is a potent inducer of hepatic enzymes involved in the detoxification of chemical carcinogens and merits further investigation for chemopreventive activity. © 2011 Springer-Verlag.

A group of 19 acyclic terpenes have been evaluated for potential toxicity/carcinogenicity by molecular orbital determinations of their spatial and electronic parameters, and hence prediction of their metabolic activation or detoxication by the cytochrome P-450 (CYP) superfamily of mixed-function oxidase enzymes. Previous studies have characterized the spatial dimensions of the CYP1A1, 1A2 and 2E1 enzymes, which are known to activate mutagens and carcinogens and to be involved in other mechanisms of toxicity. None of the terpenes was found to have shape or electronic parameters appropriate for metabolic activation by CYP1A1 or 1A2, and hence they are unlikely to be carcinogenic or mutagenic. Furthermore, none of these chemicals had spatial parameters critical for substrates of CYP2E, and they are therefore unlikely to induce the formation of reactive oxygen species (ROS) or to initiate or promote malignancy or toxicity by mechanisms involving ROS. However, citral, and others of these terpenes, are known to undergo metabolism to carboxylic acids that may induce CYP4, and are therefore possible inducers of hepatic peroxisomal proliferation at high dosage, which may have implications for possible hepatotoxicity.

The toxicity and carcinogenicity of drugs and other chemicals is, in most cases, mediated by highly reactive intermediates which are generated following metabolism catalysed by the enzymic apparatus of the exposed organisms. These reactive intermediates readily interact covalently with vital cellular components to provoke toxicity and carcinogenicity. The ubiquitous cytochrome P450-dependent mixed-function oxidases are the most important enzyme system in the activation of chemicals. This enzyme system comprises a number of families, each of which contains one or more subfamilies. The CYPIA and CYP2E subfamilies are the most closely associated with the production of reactive intermediates and, consequently, the manifestation of toxicity and carcinogenicity. A computer based molecular structure procedure (COMPACT) has been developed which, via a calculation of the molecular and electronic structure of the chemical, determines whether the chemical will interact with either of these two cytochrome P450 subfamilies and hence be metabolised to form reactive intermediates that manifest toxicity. As the basal levels of these two subfamilies are generally low, the ability of a chemical to induce them selectively, on repeated administration, is an important determinant of its toxic and carcinogenic potential. This inductive capability may be determined in short-term studies (ENACT) using only a small number of animals. Thus the combination of COMPACT and ENACT provides a rapid and inexpensive means for the preliminary screening of chemicals for toxicity and carcinogenicity before undertaking the long-term and expensive rodent lifetime bioassays.

The present studies were undertaken to provide a rationale for the observation that benzo( a )pyrene and 2-acetylaminofluorene induce the hepatic CYP1 A1 protein, whereas their non-carcinogenic isomers benzo( e )pyrene and 4-acetylaminofluorene are, at best, relatively very weak inducers. Using amino acid sequence alignment, a molecular model of the CYP1A1 was constructed by analogy to CYP101, the bacterial protein for which the 3-dimensional structure is known from X-ray crystallographic analysis. The putative structure of the active site of the CYP1A1 protein shows the presence of two phenylalanine residues preferentially aligned in parallel orientation, presumably functioning as a ‘sieve’ for planar molecules, the established substrates of CYP1A1. The molecular dimensions of this putative access channel show a width and depth of 8.321 and 3.261 A, respectively. The width of 4-acetylaminofluorene, 8.794 A, and benzo( e )pyrene, 9.153 A, precludes their passage through this channel access in contrast to benzo( a )pyrene and 2-acetylaminofluorene having a width of 7.150 and 5.283 A, respectively, explaining their difference in CYP1A1 induction potential.

The effects of long-term insulin-dependent diabetes on the enzymatic activities of hepatic cytochrome P450 isozymes were determined in rats rendered diabetic by the administration of streptozotocin and killed 4, 8, and 12 weeks following treatment. The O-dealkylations of ethoxyresorufin and pentoxyresorufin were elevated in the diabetic animals throughout the study, the extent of increase being similar at all three time points. p-Nitrophenol hydroxylase activity was induced in the diabetic animals 4 weeks following treatment with streptozotocin, but the extent of increase became less pronounced with the progress of the disease. A modest increase in ethylmorphine N-demethylase activity was also observed but only in the diabetic animals killed 4 weeks after the induction of diabetes. Finally, lauric acid hydroxylase activity was elevated in the diabetic animals 4 weeks following streptozotocin administration but then declined rapidly with the duration of the disease. It is concluded that the duration of diabetes modulates the hepatic cytochrome P450 profile, with the effect being isoenzyme specific. Mechanisms that may account for these changes are discussed.

(1994). Molecular Modeling of Enzymes and Receptors Involved in Carcinogenesis: Qsars and Compacted-3D. Drug Metabolism Reviews: Vol. 26, No. 1-2, pp. 261-285.

1. Ferret on a high fat diet accumulated large amounts of fat in its liver and had blood acetoacetate and beta-hydroxybutyrate concentrations 250 and 375% of those in control animals. 2. The high fat diet alone increased ferret liver microsomal 7-ethoxyresorufin O-deethylase (EROD) activity by 90%, but had no effect on 7-methoxy-, 7-pentoxy-, or 7-benzyloxy-resorufin, O-dealkylase activities. Administration of 3-methylcholanthrene (MC) increased only liver EROD activity, by 5- to 6-fold, in ferret on both high fat and control diets. Induction of EROD, but not MROD activity, in ferret on the high fat diet indicates that P4501A1, but not P4501A2, is induced. 3. Activation of 3H-paracetamol, measured by covalent tissue binding to ferret liver microsomal fractions, was increased three-fold in ferret on the high fat diet, nine-fold by MC administered to ferret on a control diet, and 13-fold by MC given to ferret on the high fat diet. Similar results were obtained with activation of the cooked-food amine, Glu-P-1, by ferret liver microsomes. 4. Western blots with antibodies to rat liver P450s showed that ferret liver contains proteins orthologous with rat liver P4504A1 and bifunctional protein. However, whereas clofibrate, similar to high fat diets, induced these two proteins in rat liver, no increase of these proteins occurred in liver of ferret fed a high fat diet. Western blots also showed that ferret liver contains no P4501A1 or 1A2, and although these two proteins were induced by MC, no induction occurred when ferret was fed the high fat diet alone. Ferret liver microsomes also contain a protein recognized by rat anti-P4502E1 but of a lower molecular weight. 5. Immunosorbent (ELISA) analyses of ferret liver for P4501A1 and 4A1 showed that the high fat diet increased a protein orthologous to rat P4501A1 but did not increase any protein orthologous to rat P4504A1. 6. These findings indicate that the high fat diet does not induce ferret liver bifunctional protein or P4504A1 enzyme protein, but may enhance liver P4501A1 and 1A2 activities through the hyperketonaemia resulting from the high dietary fat. The conflicting P450 results, namely Glu-P-1 activation but no MROD activity for P4501A2, high EROD activity and ELISA quantification of P4501A1, but no positive Western blot, are probably due to differences in substrate specificity and immunological characteristics between rat and ferret enzymes.

In the present study aqueous extracts of green tea, at the concentrations customarily consumed by humans, were evaluated for their antimutagenic activity against major classes of dietary and occupational carcinogens. Green tea extracts caused a very marked and concentration-dependent inhibition of the Aroclor 1254-hepatic S9-mediated mutagenicity of heterocyclic amines (IQ and Glu-P-1) and polycyclic aromatic hydrocarbons (benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene) and of the isoniazid-induced S9-mediated mutagenicity of nitrosamines (nitrosopiperidine and nitrosopyrrolidine). Similar inhibition was seen in the mutagenicity of the two aromatic amines, namely 2-amino-fluorene and 2-aminoanthracene, whether Aroclor 1254-S9, isolated microsomes or cytosol served as the activation system. Finally, the mutagenicity of the direct-acting mutagens 9-aminoacridine and MNNG was also suppressed by green tea extracts, but the effect was less pronounced when compared with the indirect-acting mutagens. Green tea extracts caused a marked and concentration-dependent decrease in the O-dealkylation of methoxyresorufin, ethoxyresorufin and pentoxyresorufin. A similar inhibition of the NADPH-dependent reduction of cytochrome c was also observed. Following the termination of the microsomal metabolism of the various promutagens, incorporation of green tea extracts into the activation system resulted in a comparatively modest inhibition of their mutagenic response. It is concluded that aqueous extracts of green tea possess marked antimutagenic potential against a variety of important dietary and environmental mutagens. Two mechanisms appear to be responsible. The first involves a direct interaction between the reactive genotoxic species of the various promutagens and nucleophilic tea component(s) present in the aqueous extracts.(ABSTRACT TRUNCATED AT 250 WORDS)

A study has been undertaken to identify the properties of 2-(acetylamino)fluorene which render it carcinogenic, in contrast to the 4-isomer, the carcinogenic potential of which is, at best, very weak. Compared to the 4-isomer, 2-(acetylamino)fluorene was a more potent inducer of cytochrome P450 1A (P450 1A) activity, as exemplified by the O-deethylation of ethoxyresorufin (P450 1A1), the metabolic activation of Glu-P-1 (P450 1A2), and immunoblot analysis. These findings were consistent with the relative affinity of these compounds for the cytosolic aromatic hydrocarbon receptor, determined by the displacement of tetrachlorodibenzo-p-dioxin. In the presence of Aroclor 1254-induced hepatic microsomal and cytosolic preparations 2-(acetylamino)-fluorene elicited a potent mutagenic response in the Ames test whereas the 4-isomer, in both cases, elicited a weak mutagenic response. Molecular orbital calculations of the relative energetics of the nitrenium ions revealed that the ion of the 2-isomer was more stable than the nitrenium ion of 4-(acetylamino)fluorene. Control hepatic microsomal and cytosolic fractions could activate 2-(acetylamino)fluorene to mutagens in the Ames test. Pretreatment of animals with 2-(acetylamino)fluorene enhanced both the microsome- and cytosol-mediated activation. In contrast, microsomal and cytosolic fractions from control animals could not activate 4-(acetylamino)fluorene, but following pretreatment with the compound itself, a weak mutagenic response in the presence of microsomes, but not of cytosol, was evident.(ABSTRACT TRUNCATED AT 250 WORDS)

The currently accepted view is that the chemopreventive activity of glucosinolates is exclusively mediated by their degradation products, such as isothiocyanates. In the present study, evidence is presented for the first time that intact glucosinolates can modulate carcinogen-metabolising enzyme systems. The glucosinolates glucoraphanin and glucoerucin were isolated from cruciferous vegetables and incubated with precision-cut rat liver slices. Both glucosinolates elevated the O-dealkylations of methoxy- and ethoxyresorufin, markers for CYP1 activity; supplementation of the incubation medium with myrosinase, the enzyme that converts glucosinolates to their corresponding isothiocyanates, abolished these effects. Moreover, both glucoerucin and glucoraphanin increased the apoprotein levels of microsomal CYP1A1, CYP1A2 and CYP1B1. At higher concentrations, both glucosinolates enhanced quinone reductase activity, whereas glucoraphanin also elevated glutathione S-transferase; in this instance, however, supplementation of the incubation medium with myrosinase exacerbated the inductive effect. Finally, both glucosinolates increased modestly cytosolic quinone reductase, GSTα and GSTμ protein levels, which became more pronounced when myrosinase was added to the incubations with the glucosinolate. It may be inferred that intact glucosinolates can modulate the activity of hepatic carcinogen-metabolising enzyme systems and this is likely to impact on the chemopreventive activity linked to cruciferous vegetable consumption.

The cytochromes P-450 detoxicate most chemicals, but activate carcinogens and other toxic chemicals by oxygenating them to reactive intermediates. A new strategy based on the P-450s has resulted in the development of a computer program (COMPACT) and of a programme of enzyme induction studies, as short-term tests to predict the potential toxicity/carcinogenicity of chemicals. Most carcinogens are metabolically activated by P450 I (P-448); P450 IIE and P450 IV also mediate chemical toxicity by producing oxygen radicals. P450 IIB mostly results in detoxication, but where chemicals are oxygenated with difficulty (e.g. phenobarbitone), the cytochrome is induced and futile cycling and oxygen radical formation may result. Oxygen radicals are highly toxic, mutagenic and carcinogenic and mediate much chemical toxicity in small rodents. As P450 genes of man differ from those of rodents, the latter have limited value as surrogates for man in chemical safety evaluation; many short-term tests, such as the Ames test, although valuable in detecting genotoxic chemicals, are similarly limited by their rat liver microsomal activation (S-9) systems. COMPACT is able to overcome both of these limitations, requires no laboratory animals or tissues, and can be conducted from a knowledge of the structure of the chemical, even prior to its synthesis. As the rodent tumorigenicity assay and the Ames test are currently regarded as the standard procedures for safety evaluation, COMPACT was validated against these in a study of 100 miscellaneous chemicals and showed excellent correlations. A quantitative structure-activity relationship (QSAR) for a series of 14 methyl benzanthracenes shows good correlation of mutagenicity with the energy of the lowest empty molecular orbital [E(LEMO)] values of the chemicals. A strategy for COMPACT and P-450 induction studies in chemical safety evaluation is presented.

The ability of the plant phenol ellagic acid to inhibit the mutagenicity of the food mutagen IQ was evaluated using Salmonella typhimurium strain TA98 in the Ames mutagenicity test. Ellagic acid caused a concentration-dependent decrease in the S-9- and microsome-mediated mutagenicity of IQ. The plant phenol did not interact directly with the IQ-derived mutagenic species and did not modify the cytosol-mediated activation of the promutagen. At the concentrations used in the mutagenicity studies, ellagic acid failed to inhibit microsomal mixed-function oxidase activity, including that mediated by the P450I family responsible for the bioactivation of IQ, despite being an essentially planar molecule as indicated by computer-graphic analysis. The inhibitory effect of ellagic acid was independent of its ability to chelate Mg2+. However, pre-incubation of ellagic acid with the bacteria, followed by removal of the plant phenol, did not completely prevent the inhibitory effect of the phenol on the mutagenicity of IQ. Intraperitoneal administration of ellagic acid to rats caused a decrease in total cytochrome P-450 levels and related activities as well as in cytosolic glutathione S-transferase activity. Finally, the possibility that the reported anticarcinogenic action of ellagic acid reflects nothing more than non-selective destruction of hepatic cytochromes P-450, and thus reduced chemical activation, is considered.

The effect of non-insulin-dependent diabetes on the hepatic microsomal cytochrome P450-dependent mixed-function oxidase system and on cytosolic glutathione S-transferase activity was determined using the spontaneously obese-diabetic (ob/ob) mouse model. The activities of the xenobiotic-metabolizing cytochrome P450 proteins were monitored by the use of chemical probes. Non-insulin-dependent diabetes did not influence the hepatic metabolism of substrates associated with the P450 I, IIB, IIE, III and IV families of cytochromes. In contrast, cytosolic glutathione S-transferase activity was markedly reduced and glutathione levels were significantly lowered. These findings raise the possibility that patients suffering from this disease may be more susceptible to chemicals that rely on glutathione conjugation for their deactivation.

1. To evaluate the role of ketone bodies in diabetes-induced changes in hepatic cytochrome P450 composition, rats were treated with acetone, 3-hydroxybutyrate or 1,3-butanediol. 2. Treatment with acetone enhanced the rat hepatic O-dealkylations of ethoxyresorufin and methoxyresorufin, and the hydroxylation of p-nitrophenol, but had no effect on lauric acid hydroxylation and ethylmorphine N-demethylation. Neither 3-hydroxybutyrate nor 1,3-butanediol modulated the metabolism of the above substrates. 3. Immunoblot analysis of hepatic microsomal proteins revealed that treatment with acetone increased the apoprotein levels of P4501A2, P4502B1/2 and P4502E1. 4. It is concluded that acetone is responsible, at least partly, for the diabetes-induced increase in hepatic microsomal P4501A2, P4502B1/2 and P4502E1 proteins but does not mediate the increases in the P4503A1 and P4504A1 proteins. On the basis of work from our own and other laboratories a mechanism for the diabetes-induced changes in hepatic cytochrome P450 proteins is proposed.

On the basis of studies conducted in animals, it has been established that isothiocyanates suppress cytochrome P450 activity, leading to impairment of the bioactivation of carcinogens, this being a principal mechanism of their chemopreventive activity. However, no studies have been carried out in human tissue to ascertain whether hepatic cytochrome P450 composition is similarly modulated, and this is the objective of the present studies. Precision-cut liver slices from four donors were incubated with a range of concentrations of phenethyl isothiocyanate (PEITC) for 24 h, and the expression and activity of cytochrome P450 enzymes were determined; similar studies were performed in rat slices for comparison. PEITC suppressed the O -dealkylation of methoxyresorufin in all human livers and this was accompanied by a parallel drop in CYP1A2 apoprotein levels; the same effect was noted in rat liver slices. The O -dealkylation of ethoxyresorufin was also impaired in the human livers, despite a rise in CYP1A1 apoprotein levels. The CYP3A-mediated benzyloxyquinoline dealkylation was inhibited by PEITC in only two of the four human donors, whereas a rise in CYP3A4 apoprotein levels was noted in all human livers, albeit to different extent. It is concluded that: (a) PEITC can modulate cytochrome P450 composition in human liver, and (b) PEITC, at concentrations that can be achieved by dietary intake, can antagonise the carcinogenicity of chemicals which rely on the CYP1 family for their bioactivation such as heterocyclic amines and polycyclic aromatic hydrocarbons, and this is likely to be a major contributory mechanism to its chemopreventive activity.

Isothiocyanate up-regulation of hepatic NAD(P)H:quinone oxidoreductase (NQO1) and glutathione S-transferases (GSTs) is an integral mechanism of their chemoprevention. In this paper, for the first time, the potential of the isothiocyanates erucin and sulforaphane to modulate these enzymes was investigated in two human livers and compared to rat liver. Precision-cut liver slices were incubated with erucin or sulforaphane (1-50 microM). Both isothiocyanates elevated NQO1 activity in rat slices that was paralleled by a fourfold rise in protein levels. No change in activity was noted in human slices, and only a weak rise in protein levels, < 10% of that in rat, was observed in only one of the human livers, whereas the other was refractive. GST activity, assessed with three substrates, was elevated in rat slices treated with either isothiocyanate, and was accompanied by a rise in GSTalpha and GSTmicro, but not GSTpi, protein levels. A rise in activity and in GSTalpha and GSTmu protein levels was also noted in one of the human livers. It appears that erucin and sulforaphane elevate GST expression in isoform-specific manner in both rat and human liver, whereas NQO1 is inducible by these compounds only in rat liver and very poorly in human liver.

Administration of the non-steroidal anti-inflammatory drug benoxaprofen to rats gave rise to significant increases in the hepatic O-dealkylations of ethoxyresorufin and methoxyresorufin and in the 12-hydroxylation of lauric acid but, in contrast, the N-demethylation of dimethylnitrosamine was inhibited. Immunoblot studies employing solubilized microsomes from benoxaprofen-treated rats revealed that benoxaprofen increased the apoprotein levels of P450 IA1 and A2 and of P450 IVA1. The same treatment with benoxaprofen increased the beta-oxidation of palmitoyl CoA determined in liver homogenates, and immunoblot analysis showed an increase in the apoprotein levels of the trans-2-enoyl CoA hydratase bifunctional protein. It is concluded that benoxaprofen is a peroxisomal proliferator which selectively induces the hepatic cytochrome P450 I and IV families. The possible implications of these findings to the well-known hepatotoxicity of this drug are discussed.

The roles of the cytochromes P450 are reviewed, with emphasis on their involvement in the detoxication of drugs and chemicals, the activation of carcinogens, and the toxicity of drugs. Cytochromes P450 have different characteristics. P450I mostly activates carcinogens and other chemicals by forming oxygenated reactive intermediates, which are also associated with the formation of neoantigens and immunotoxicity. P450IIE has a propensity to form oxygen radicals, which are cytotoxic and carcinogenic; other cytochromes generate oxygen radicals by futile cycling when activated by difficultly metabolized substrates. Novel procedures for the safety evaluation of chemicals are described; COMPACT is based on the computer graphic determination of the spatial conformation and electronic structure of chemicals to enable their activating cytochromes P450, and hence their toxicity, to be established; ENACT is based on quantifying the induction of individual cytochromes P450, since the extent of induction of P450I, and possibly other activating cytochromes, is directly related to the carcinogenic potential of the chemical.Key words: cytochromes P450, oxygen radicals, enzyme induction, drug metabolism, chemical toxicity.

On the basis of animal studies, the chemopreventive activity of isothiocyanates has been linked to their ability to modulate carcinogen-metabolising enzyme systems, including cytochrome P450. However, the potential of isothiocyanates to influence these enzyme systems in human liver has not been investigated. We have evaluated the modulation of cytochrome P450 expression in two human liver samples by erucin and sulforaphane, in comparison to rat, following the incubation of precision-cut human and rat liver slices with the two isothiocyanates. Both compounds failed to influence cytochrome P450 activity, as exemplified by the dealkylations of methoxy-, ethoxy- and pentoxyresorufin, and benzyloxyquinoline, in either human or rat liver. Impairment of activity was, however, observed in some activities at high concentrations (50microM), which was attributed to toxicity. At the apoprotein level, however, both compounds markedly elevated CYP1A2/1B1 levels in rat liver, but in human liver only a modest increase was evident, and only in one of the livers. CYP3A2 apoprotein levels were modestly elevated in rat liver by both isothiocyanates both of which, however, failed to influence CYP3A4 expression in human liver. Neither isothiocyanate, in either rat or human liver, modulated CYP2B apoprotein levels. It may be inferred that (a) human and rat liver differ in their response to erucin and sulforaphane, (b) erucin and sulforaphane, despite being small molecular weight aliphatic compounds, up-regulate the CYP1 family but no increase in activity is observed as a result of mechanism-based inhibition, and (c) the chemopreventive effect of isothiocyanates, at dietary levels of intake, is unlikely to be due to inhibition of the cytochrome P450-mediated bioactivation of carcinogens.

The cytosolic glutathione S-transferases (GSTs) comprise a pivotal enzyme system protecting the cell from electrophilic compounds. It plays a major role in the detoxication of the primary and dihydrodiol epoxides of polycyclic aromatic hydrocarbons (PAHs), so that modulation of this enzyme system by PAHs will impact on their carcinogenic activity. The potential of six structurally diverse PAHs, namely benzo[a]pyrene (B[a]P), fluoranthene, benzo[b]fluoranthene (B[b]F), dibenzo[a,l]pyrene, dibenzo[a,h]anthracene (D[a,h]A) and 1-methhylphenanthrene, to modulate hepatic GST activity was investigated in human precision-cut slices and compared to rat slices, a species frequently used in long-term carcinogenicity studies; changes were monitored at the activity, using three different substrates, protein and mRNA levels. When activity was monitored using the alpha-class selective 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, B[b]F was the only PAH that caused an increase in activity, which was accompanied by a rise in the Ya immunoreacting band. In rat slices, in addition to B[b]F, B[a]P and D[a,h]A also enhanced activity, being paralleled with increased levels of the Ya immunoreacting band. In the rat, all PAHs elevated mRNA levels. In both human and rat liver slices, only B[b]F enhanced activity when 1-chloro-2,4-dinitrobenzene (CDNB) served as substrate. To investigate tissue differences, similar studies were undertaken in precision-cut rat lung slices, incubated with PAHs under identical conditions, using CDNB, as this was the only substrate for which activity could be detected; none of the PAHs studied stimulated activity. It is concluded that some PAHs have the potential to induce GST activity in human liver tissue and that species and tissue differences exist in the induction of this enzyme system in the rat. However, the extent of induction of GST activity is very modest compared with the effect these compounds have on CYP1 expression, the family responsible for their bioactivation, and it is unlikely to compensate for the enhanced production of reactive intermediates.

PART A: Chapter 1: Cytochrome P450 Structure and Function: An Evolutionary Perspective Chapter 2: Generation of reactive intermediates by cytochromes P450 PART B: Chapter 3: The CYP1A subfamily Chapter 4: The CYP1B subfamily Chapter 5: The CYP2A subfamily Chapter 6: The CYP2B subfamily Chapter 7: The CYP2C subfamily Chapter 8: The CYP2D subfamily Chapter 9: The CYP2E subfamily Chapter 10: The CYP2F, CYP2G and CYP2J subfamilies Chapter 11: The CYP3 family Chapter 12: The CYP4 family PART C: Chapter 13: Receptor-mediated regulation of cytochromes P450 Chapter 14: Modulation of cytochrome P450 activity by phytochemicals Chapter 15: Cytochromes P450 in cancer therapeutics

The molecular and electronic structural characteristics of the hepatotoxic and phototoxic antirheumatic drug, benoxaprofen, indicate that it falls in the interface between the area of parametric space associated with substrates of cytochrome P450I and that associated with substrates of other cytochromes P450, combining fairly planar molecular geometry (area/depth2 = 2.5) with relatively low activation energy (ΔE = E(LEMO) − E(HOMO) = 12.0). Benoxaprofen may therefore be a substrate for cytochrome P450I so that, like many other P450I substrates, it may be oxygenated to a reactive intermediate, thereby causing hepatotoxicity. Benoxaprofen also has a molecular structure closely similar to that of clofibrate and may thus be a possible substrate for cytochrome P450IV and result in hepatic peroxisomal proliferation. The structural similarity of benoxaprofen with the furocoumarin, psoralen, is associated with its known phototoxicity. QSAR analysis of the acute toxicities and anti-inflammatory activities of 16 analogues of benoxaprofen has been undertaken to identify a drug candidate likely to have similar anti-inflammatory activity to benoxaprofen but with lower toxicity.

(1990). The Cytochrome P450 I Gene Family of Microsomal Hemoproteins and Their Role in the Metabolic Activation of Chemicals. Drug Metabolism Reviews: Vol. 22, No. 1, pp. 1-85.

The hypothesis has been put forward that genotoxic aromatic amines which induce the P450 I family of haemoproteins, the major enzyme involved in their bioactivation, are more likely to be carcinogenic when compared to those chemicals that fail to do so. Induction of the hepatic P450 I family of proteins by carcinogenic aromatic amines and their non-carcinogenic isomers and analogues was investigated in the rat and correlated to their carcinogenic potential. The activity of the P450 I A1 protein was monitored by the O-deethylation of ethoxyresorufin and of the P450 I A2 by the activation of the premutagen Glu-P-1 to mutagenic intermediates in the Ames test. Results were always confirmed immunologically in Western blots employing antibodies to rat P450 I A1 which recognize both proteins of the P450 I family. With all groups of chemicals used in the present study, the members displaying carcinogenicity were always the more potent inducers, while the non-carcinogenic isomers or analogues displayed little or no induction. It appears that a relationship exists between the carcinogenicity of aromatic amines and their ability to induce hepatic P450 I activity.

The hypothesis has been put forward that mutagenic polycyclic aromatic hydrocarbons which induce the P-450 I family of cytochromes, the major enzyme system responsible for their activation, are likely to be carcinogenic. In order to test this hypothesis, rats have been pretreated with a number of polycyclic aromatic hydrocarbons of different mutagenic and carcinogenic potency and hepatic P-450 I activity was monitored using chemical probes such as the O-deethylation of ethoxyresorufin and metabolic activation of Glu-P-1 to mutagens, and immunologically employing polyclonal antibodies against purified rat P-450 I A1. All compounds studied enhanced P-450 I activity and induced P-450 I apoproteins but the extent of induction was very markedly different. The results are discussed with reference to the mutagenicity of these chemicals in the Ames test and their carcinogenicity in the classical mouse skin model. A relationship appears to exist between carcinogenicity of polycyclic aromatic hydrocarbons and their ability to induce hepatic P-450 I activity.

A number of enzyme systems participate in the metabolism of chemicals, but undoubtedly the most important are the cytochromes P450 (CYP). It is a versatile enzyme system, capable of metabolising structurally diverse chemicals. To achieve this broad substrate specificity it exists as a superfamily of enzymes, each family being characterised by different substrate specificity; families CYP1, CYP2 and CYP3 are responsible for the metabolism of exogenous chemicals. Although our current knowledge of the expression and function of cytochromes P450 in humans and laboratory animals is extensive, little is known about this enzyme system in food-producing animals, despite its dominant role in the metabolism of veterinary drugs, and the crucial role it plays in controlling the levels of drug and other chemical residues in edible tissues and food products that humans consume, a matter of major current concern. Most studies dealing with the expression of cytochromes P450 in food-producing animals utilised substrate probes defined in rats and humans and/or antibodies raised to rat or human antigens. Such an approach can prove misleading as it assumes that orthologue proteins in other animals share the same substrate specificity and, moreover, although antibodies raised to human or rat antigens may recognise epitopes in other species, they do not constitute unequivocal proof that the detected proteins are structurally identical. Despite these drawbacks, there is substantial experimental evidence that CYP1, CYP2 and CYP3 families are expressed in food-producing animals, but their role in the metabolism of veterinary drugs and other xenobiotics has not been addressed.

The objective of the present study was to evaluate the stability of cytochrome P450 enzymes and of the conjugation enzyme systems epoxide hydrolase, glucuronosyl transferase, sulphotransferase and glutathione S-transferase in precision-cut rat lung slices incubated in RPMI media for different time periods up to 72 h. Moreover, the effect of culturing of lung slices on total glutathione levels and glutathione reductase was also investigated. Monitoring of cytochrome P450 activity was achieved using established diagnostic probes, but when activity in the lung was low the maintenance of the various enzymes in culture was determined immunologically using Western blotting. The dealkylation of pentoxyresorufin declined markedly during the first 4 h of incubation but in the case of ethoxyresorufin loss of activity was more gradual and less severe. Western blot analysis revealed that the rate of decrease in cytochrome P450 apoprotein levels was isoform-specific with CYP2E1 being the most stable and CYP3A the least stable. Generally, phase II activities, especially cytosolic sulphotransferase, were relatively more stable throughout the incubation period compared with cytochromes P450. Finally, glutathione reductase activity and total glutathione levels were maintained throughout the 72 h incubation. The present studies indicate that xenobiotic-metabolising enzymes in precision-cut rat lung slices decline in culture, but the rate of loss differs and depends on the nature of the enzyme.