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Neurodegenerative diseases (NDDs) are chronic conditions that have drawn robust interest from the scientific community. Phytotherapeutic agents are becoming an important source of chemicals for the treatment and management of NDDs. Various secondary metabolites have been isolated from Melicope lunu-ankenda plant leaves, including phenolic acid derivatives. However, their neuroprotective activity remains unclear. Thus, the aim of this study is to elucidate the in vitro neuroprotective activity of 7-geranyloxycinnamic acid isolated from Melicope lunu-ankenda leaves. The neuroprotective activity was evaluated in differentiated human neuroblastoma (SH-SY5Y) cells by monitoring cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Moreover, the potential to impair apoptosis in differentiated cells was investigated employing the Annexin V-FITC assay, acridine orange and propidium iodide (AO/PI) staining, and fluorescence microscopy. Morphological assessment and ultrastructural analysis were performed using scanning and transmission electron microscopy to evaluate the effect of 7-geranyloxycinnamic acid on surface morphology and internal features of the differentiated cells. Pre-treatment of neuronal cells with 7-geranyloxycinnamic acid significantly protected the differentiated SH-SY5Y cells against H2O2-induced apoptosis. Cytoskeleton and cytoplasmic inclusion were similarly protected by the 7-geranyloxycinnamic acid treatment. The present findings demonstrate the neuroprotective potential of 7-geranyloxycinnamic acid against H2O2-induced neurotoxicity in neuronal cells, which is an established hallmark of neuronal disorders.

Gluconasturtiin, a glucosinolate present in watercress, is hydrolysed by myrosinase to form gluconasturtiin-isothiocyanate (GNST-ITC), which has potential chemopreventive effects, however, the underlying mechanisms of action have not been explored, mainly in human cell lines. The purpose of the study is to evaluate the cytotoxicity of GNST-ITC and to further assess its potential to induce apoptosis. GNST-ITC inhibited cell proliferation in both human hepatocarcinoma (HepG2) and human breast adenocarcinoma (MCF-7) cells with IC50 values of 7.83 &micro, M and 5.02 &micro, M, respectively. Morphological changes as a result of GNST-ITC-induced apoptosis showed chromatin condensation, nuclear fragmentation, and membrane blebbing. Additionally, Annexin V assay showed proportion of cells in early and late apoptosis upon exposure to GNST-ITC in a time-dependent manner. To delineate the mechanism of apoptosis, cell cycle arrest and expression of caspases were studied. GNST-ITC induced a time-dependent G2/M phase arrest, with reduction of 82% and 93% in HepG2 and MCF-7 cell lines, respectively. The same treatment also led to the subsequent expression of caspase-3/7 and -9 in both cells demonstrating mitochondrial-associated cell death. Collectively, these results reveal that GNST-ITC can inhibit cell proliferation and can induce cell death in HepG2 and MCF-7 cancer cells via apoptosis, highlighting its potential development as an anticancer agent.

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.

Introduction Electronic cigarettes (EC) represent a safer alternative to tobacco, however, there is limited information on adverse health effects, especially after long term use. Among potential harmful constituents of EC aerosols nicotine is the major addictive chemical and is a precursor for carcinogenic tobacco-specific nitrosamines (TSNAs). Multiple studies have highlighted the ability of nicotine to compromise blood brain barrier (BBB) integrity, altering transport and receptor systems. One knowledge gap is the effect of e-fluids on enzymes responsible for metabolism of toxicants including carcinogens, Therefore, the aim of this study was to examine the effect of nicotine and e-fluid on the expression of cytochrome P450 enzymes (CYPs) in an in vitro model of human BBB, the hCMEC/D3 cell line. Methods The hCMEC/D3 cells were used to investigate time and dose-dependent responses of nicotine or e-fluid and EC aerosol condensate (normalised to nicotine concentration). High quality RNA was extracted and analysed by qRT-PCR for the expression of genes encoding enzymes involved in nicotine metabolism and bioactivation of procarcinogens (CYP1A1, CYP1A2, CYP2A6, CYP2E1, CYP2A13, CYP3A4) and recently described extrahepatic CYP isoforms (CYP2U1 and CYP2S1), which are highly expressed in BBB. Results qRT-PCR results were normalised using UBC reference gene and expressed as a fold change between non treated and treated cells (n=3, p

Introduction Use of electronic cigarettes (ECs) has increased rapidly since they were introduced in Europe in 2006 especially in the UK. Claims have been made for a role in tobacco harm reduction and smoking cessation. Key to these claims is the ability of EC to maintain nicotine levels to limit cravings, withdrawal and nicotine dependence. Therefore, the aim of the current study is to monitor nicotine intake in heavy smokers (>10 cig/day, n=13 completed) transitioning from cigarette smoking to EC-only use whilst collecting psychometric data on nicotine dependence (Fagerstrom), withdrawal (Minnesota), anxiety, depression and sleep quality. Material and Methods Urine and saliva samples as well as psychometric questionnaire data were collected at baseline (visits 1 and 2) and at days 3, 7, 21 and 28 following start of EC use. An UHPLC-HESI-HRMS method was developed, validated and applied to the determination of nicotine and its main metabolite cotinine in urine and saliva. Results The baseline median (interquartile range) urinary nicotine and cotinine levels were determined as 1534 ng/mg creatinine (317-2478) and 2082 ng/mg creatinine (1727-3632), whereas salivary nicotine and cotinine levels were 145 ng/ml (26-265) and 329 ng/ml (198-440), respectively. One-way repeated measures Anova demonstrated a significant decrease in urinary nicotine and cotinine concentration from baseline at 3 post-transition visits. A significant reduction was also observed in salivary nicotine levels on days 3 and 21 after EC transition and in salivary cotinine levels at each post-transition visit compared to baseline. Both urge and strength of urges to smoke were significantly decreased by 28days compared to baseline. Conclusions These findings offer support that EC can be effective in reducing nicotine consumption and cravings for cigarettes, suggesting the potential use of EC as a smoking cessation tool. However, the inability to achieve levels of nicotine comparable to smokers may contribute to relapse. Funding Statement Funded by European Commission Horizon 2020 programme.

Glucoraphenin, a glucosinolate present in large quantities in radish is hydrolysed by myrosinase to form the isothiocyanate sulforaphene, which is believed to be responsible for its chemopreventive activity, however, the underlying mechanisms of action have not been investigated, particularly in human cell lines. The aim of the study is to assess the cytotoxicity of sulforaphene in HepG2 cells and evaluate its potential to enhance apoptosis. The cytotoxicity of sulforaphene in HepG2 cells was carried out ensuing an initial screening with two other cell lines, MFC-7 and HT-29, where sulforaphene displayed highest toxicity in HepG2 cells following incubation at 24, 48 and 72 h. In contrast, the intact glucosinolate showed no cytotoxicity. Morphological studies indicated that sulforaphene stimulated apoptosis as exemplified by cell shrinkage, blebbing, chromatin condensation, and nuclear fragmentation. The Annexin V assay revealed significant increases in apoptosis and the same treatment increased the activity of caspases -3/7 and -9, whereas a decline in caspase-8 was observed. Impairment of cell proliferation was indicated by cell cycle arrest at the Sub G0/G1 phase as compared to the other phases. It may be concluded that sulforaphene, but not its parent glucosinolate, glucoraphenin, causes cytotoxicity and stimulates apoptosis in HepG2 cells.

Introduction The aim of this study was to quantify levels of carcinogenic tobacco specific nitrosamines (TSNAs) in human volunteers transitioning from tobacco use to electronic cigarettes (EC). ECs have increased in popularity, with consumers using ECs to aid with smoking cessation, reduce cigarette consumption, or to minimise withdrawal symptoms when smoking but not vaping is prohibited. There has been little investigation into the long term health impacts of ECs, but they are generally considered less toxic than conventional tobacco cigarettes. This is due to the absence or decreased levels of harmful chemicals commonly found in tobacco cigarettes. However, some studies have shown the presence of traces of TSNAs in e-liquid, and the endogenous nitrosation of nicotine to TSNAs in nicotine replacement therapies (NRT). These TSNAs include NNK, NNN, and NNAL, which are potent oesophageal and pulmonary carcinogens. Therefore, inhalation of these compounds may provide an added risk to the use of ECs, when compared to other NRT products. Methods Biofluids have been analysed for TSNAs in the urine and saliva of heavy smokers (>10 cigarettes/day) who quit and transition to EC use for 28 days. A solid phase extraction method using TSNA specific molecular imprinted polymers was used to concentrate samples, which were subsequently analysed using UHPLC-ESI-HRMS. Results Analysis of longitudinal urine and saliva samples (n=13) has shown a decrease in TSNA levels over the period of transition from tobacco use to EC only use. Conclusions The findings are consistent with the notion that substituting conventional cigarettes with ECs significantly lowers exposure to carcinogenic TSNAs. Further work is needed to determine whether TSNAs present in urine and saliva represent continued low level exposure to TSNAs as contaminants in EC or markers of nitrosation. Funding This work is funded by Horizon 2020, and Public Health England

A strongly electronegative, bay-region analogue of the potent carcinogen 15, 16-dihydro-11-methylcyclopenta[a]phenanthren-17-one, namely 15, 16-dihydro-11-trifluoromethylcyclopenta[a]phenanthren-17-one, is mutagenic to Salmonella typhimurium TA100. Also it is metabolized at the 1, 2- and 3, 4-positions in the A-ring as well as C-15 in the D-ring to give 3, 4-dihydroxy-3, 4, 15, 16-tetrahydro-11-trifluoromethyl-cyclopenta[a]phenanthren-17-one as the only mutagenic metabolite. In these respects its behaviour is closely similar to that of the 11-methyl compound, suggesting that the electronic nature of the bay-region substituent is rather less critical than its spatial configuration in influencing metabolism to genotoxic intermediates. It remains to be seen, however, whether the trifluoromethyl compound is also a carcinogen

Possible interactions of melatonin with concurrently administered drugs were investigated in in vitro studies utilising human hepatic post-mitochondrial preparations; similar studies were conducted with rat preparations to ascertain whether rat is a suitable surrogate for human. Drugs were selected based not only on the knowledge that the 6-hydroxylation of exogenous melatonin, its principal pathway of metabolism, is mainly mediated by hepatic CYP1A2, but also on the likelihood of the drug being concurrently administered with melatonin. Hepatic preparations were incubated with either melatonin or 6-hydroxymelatonin in the presence and absence of a range of concentrations of interacting drug, and the production of 6-sulphatoxymelatonin monitored using a radioimmunoassay procedure. Of the drugs screened, only the potent CYP1A2 inhibitor 5-methoxypsoralen impaired the 6-melatonin hydroxylation at pharmacologically relevant concentrations, and is likely to lead to clinical interactions; diazepam, tamoxifen and acetaminophen (paracetamol) did not impair the metabolic conversion of melatonin to 6-sulphatoxymelatonin at concentrations attained following therapeutic administration. 17-Ethinhyloestradiol appeared not to suppress the 6-hydroxylation of melatonin but inhibited the sulphation of 6-hydroxymelatonin, but this is unlikely to result in an interaction following therapeutic intake of the steroid. Species differences in the inhibition of melatonin metabolism in human and rat hepatic post-mitochondrial preparations were evident implying that the rat may not be an appropriate surrogate of human in such studies.

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.

The objective of this study was to define CYP2D enzymes in marmoset (Callithrix jacchus) liver microsomes, both at the activity level using debrisoquine as the model substrate and at the protein level using antibodies raised to human CYP2D6. Marmoset liver microsomes were incubated with [(14)C]debrisoquine, and the structure of the generated metabolites was determined using liquid chromatography-tandem mass spectrometry and NMR. Marmoset liver microsomes were very effective in hydroxylating debrisoquine at various positions. Although 4-hydroxydebrisoquine was formed, in contrast to rat and human it was only a minor metabolite. Debrisoquine was more extensively hydroxylated in the 7, 5, 6, and 8 positions. In addition to the monohydroxylated metabolites, a dihydroxy metabolite, namely 6,7-dihydroxydebrisoquine, was identified. Finally, metabolites that had undergone ring opening were also detected but were not investigated further. Antibodies to CYP2D6 immunoreacted with protein in marmoset and human but not rat hepatic microsomes. In conclusion, we demonstrate that marmoset liver microsomes are effective in hydroxylating debrisoquine at various positions and that they contain a protein that is immunorelated to human CYP2D6.

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 principal objectives of this study were to evaluate whether exposure of rats to low doses of isothiocyanates modulates the overall metabolism of heterocyclic amine 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ), as exemplified by urinary mutagenicity, a food carcinogen, and to relate any modifications in metabolism to changes in CYP1 and glutathione S-transferase activities. Animals were exposed to isothiocyanates either for 2 wk (long-term) or 1 day (short-term), and all animals were then treated with a single oral dose of IQ, and urine was collected daily for 3 days; animals continued to receive the isothiocyanates during this period. Urinary mutagenic activity was determined using the Ames mutagenicity assay in the presence of an activation system from Aroclor 1254-treated rats. At the end of the study, animals were killed and hepatic methoxy- and ethoxyresorufin dealkylations were determined as well as glutathione S-transferase activity. All isothiocyanates studied, namely sulforaphane, erucin, and phenethyl isothiocyanate, decreased urinary mutagenic activity, implying enhanced IQ metabolism, but only after long-term intake. Changes in mutagenic activity were not related to changes of any of the enzyme activities determined. It is concluded that long-term intake of isothiocyanates may stimulate the metabolism of IQ, but this effect is not linked to changes in hepatic CYP1A2 and glutathione S-transferase activities.

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 potential of isothiocyanates to antagonize the carcinogenicity of structurally diverse chemicals has been established in animals. A feasible mechanism of action involves protecting DNA by reducing the availability of the genotoxic metabolites of chemical carcinogens by either inhibiting their generation and/or stimulating their detoxification. In vivo as well as in vitro studies conducted in rat/human primary hepatocytes and precision-cut tissue slices have revealed that isothiocyanates can impair cytochrome P450 activity, including the CYP1 family which is the most active in the bioactivation of carcinogens, by virtue of being mechanism-based inactivators. The aromatic phenethyl isothiocyanate is the most effective of those studied, whereas aliphatic isothiocyanates such as sulforaphane and erucin necessitate high doses in order to manifest such effects that may not always be achievable through the diet. In all systems studied, isothiocyanates are strong inducers of detoxification enzyme systems including quinone reductase, glutathione S-transferase, epoxide hydrolase, and UDP-glucuronosyl transferase. Indeed, in smokers phenethyl isothiocyanate intake increases the urinary excretion of inactive mercapturate metabolites of toxic chemicals present in tobacco. Glucosinolates, the precursors of isothiocyanates, have also the potential to upregulate detoxification enzyme systems, but their contribution to the cancer chemoprevention linked to cruciferous vegetable consumption remains to be evaluated.

Isothiocyanates comprise the most promising class of chemopreventive phytochemicals. They are encountered at substantial amounts in cruciferous vegetables where they exist in the form of glucosinolates. Epidemiological studies have linked consumption of isothiocyanates to low cancer incidence at a number of sites. Furthermore, laboratory studies in animal models of cancer have established that various isothiocyanates can effectively antagonise the carcinogenicity of chemicals. A principal mechanism of action of these chemicals is to modulate the metabolism of chemical carcinogens so as to favour their metabolic detoxification. In this mini review we discuss the ability of isothiocyanates to influence carcinogen-metabolising enzyme systems, such as cytochromes P450 and conjugation systems, at dietary levels of exposure.

The principal objective of this study was to evaluate whether repeated oral administration influences the pharmacokinetic behavior of the chemopreventive agent phenethyl isothiocyanate (PEITC) in rat. Animals were treated orally with 0.5, 1.0 and 5.0 mg/kg of the isothiocyanate for 4 days, and plasma levels at various times post-administration were determined by LC/MS after the first and last day. To determine absolute bioavailability, a group of animals was treated with a single (0.5 mg/kg) intravenous dose of PEITC. Following single oral dose administration, PEITC was rapidly absorbed, peak plasma concentrations being attained within the hour, and achieved an absolute bioavailability of 77%, but displayed dose-dependent pharmacokinetics, with bioavailability decreasing and clearance increasing moderately with dose; C(max) values did not rise proportionately to the dose and volume of distribution increased. At the higher doses of 1.0 and 5.0 mg/kg, repeated administration led to higher PEITC plasma C(max) concentrations and decreased plasma clearance of the isothiocyanate leading to enhanced bioavailability.

The plasma pharmacokinetic characteristics of the chemopreventive isothiocyanate sulforaphane were determined in six human volunteers following single and repeated intake of raw broccoli. Initially, an analytical method utilising LC-MS/MS, capable of determining low levels of sulforaphane in human plasma was developed and validated. The plasma profile of the isothiocyanate best fitted a two-compartment pharmacokinetic model. Sulforaphane was rapidly absorbed with peak plasma levels being attained within 1.5h, and was characterised by a long terminal elimination phase. Repeated intake of broccoli had no impact on the pharmacokinetic behaviour or plasma levels of sulforaphane, and there was no evidence of accumulation.

There is experimental and epidemiological evidence demonstrating that polycyclic aromatic hydrocarbons (PAHs) are involved in the pathogenesis of cardiovascular diseases. However, heterocyclic amines (HAs), a class of carcinogenic compounds present in food, which share many biochemical features with PAHs, have not received much attention. Previous reports have shown that the heterocyclic amine 2-amino-9H-pyrido[2,3-b]indole (AalphaC) binds and metabolically affects endothelial cells in animal models suggesting a potential role in vascular remodeling. The present study investigates the effect of exposure to HAs on atherosclerotic plaque development in the apoE(-/-) mice. We observed that animals treated with AalphaC developed atherosclerotic lesions characterized by lower lipid content but richer in inflammatory cells and collagen content when compared with control animals. Moreover, atherosclerotic plaques from AalphaC-treated apoE(-/-) mice were also smaller with a marked reduction in the tunica media thickness. Furthermore, total cholesterol levels were significantly reduced in AalphaC-treated apoE(-/-) mice. In contrast to what has been previously reported for PAHs, we provide for the first time evidence that HAs may protect against cardiovascular disease by inducing stable atherosclerotic plaques and reducing circulating cholesterol levels. These results open new avenues to further investigate the role of these food-borne carcinogens in cardiovascular physiology and pathology.

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.

Administration of dietary doses of the isothiocyanate erucin had no effect on rat hepatic cytochrome P450 activity or protein levels, but at higher doses a rise in CYP1A/B1 protein levels was evident. In lung, treatment with erucin, as well as sulforaphane, failed to modulate cytochrome P450 activities but elevated CYP1A/B1 protein levels. In liver, erucin stimulated quinone reductase activity accompanied by a rise in protein. Glutathione S-transferase activity was unaffected, but GSTalpha and GSTmu protein levels increased. In lung, both isothiocyanates increased quinone reductase paralleled by a rise in protein levels; at the higher dose both isothiocyanates elevated moderately GSTalpha levels. Hepatic microsomes converted both isothiocyanates to metabolites that impaired cytochrome P450 activity, which was antagonized by reduced glutathione. It may be concluded that erucin may protect against carcinogens by stimulating the detoxication of quinones but is unlikely to significantly influence reactive intermediate generation through modulation of cytochrome P450 activity.

Although exposure to polycyclic aromatic hydrocarbons (PAHs) occurs mostly through mixtures, hazard and risk assessment are mostly based on the effects caused by individual compounds. The objective of the current study was to investigate whether interactions between PAHs occur, focusing on gene expression (as measured by cDNA microarrays) and DNA adduct formation. The effects of benzo[a]pyrene or dibenzo[a,h]anthracene (DB[a,h]A) alone and in binary mixtures with another PAH (DB[a,h]A, benzo[b]fluoranthene, fluoranthene or dibenzo[a,l]pyrene) were investigated using precision-cut rat liver slices. All compounds significantly modulated the expression of several genes, but overlap between genes affected by the mixture and by the individual compounds was relatively small. All mixtures showed an antagonistic response on total gene expression profiles. Moreover, at the level of individual genes, mostly antagonism was evident, with additivity and synergism observed for only a few genes. As far as DNA adduct formation is concerned, the binary mixtures generally caused antagonism. The effects in liver slices suggest a lower carcinogenic potency of PAH mixtures than estimated based on additivity of individual compounds. © The Author 2008. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved.

Sulforaphane is a naturally occurring isothiocyanate with promising chemopreventive activity. An analytical method, utilising liquid chromatography-MS/MS, which allows the determination of sulforaphane in small volumes of rat plasma following exposure to low dietary doses, was developed and validated, and employed to determine its absolute bioavailability and pharmacokinetic characteristics. Rats were treated with either a single intravenous dose of sulforaphane (2·8 μmol/kg) or single oral doses of 2·8, 5·6 and 28 μmol/kg. Sulforaphane plasma concentrations were determined in blood samples withdrawn from the rat tail at regular time intervals. Following intravenous administration, the plasma profile of sulforaphane was best described by a two-compartment pharmacokinetic model, with a prolonged terminal phase. Sulforaphane was very well and rapidly absorbed and displayed an absolute bioavailability of 82 %, which, however, decreased at the higher doses, indicating a dose-dependent pharmacokinetic behaviour; similarly,Cmaxvalues did not rise proportionately to the dose. At the highest dose used, the rate of absorption constantkab, biological half-lifet½and apparent volume of distribution decreased significantly. It is concluded that in the rat orally administered sulforaphane is rapidly absorbed, achieving high absolute bioavailability at low dietary doses, but dose-dependent pharmacokinetics was evident, with bioavailability decreasing with increasing dose.

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.

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. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 764-775, 2017.

Isothiocyanates are small molecules characterized by high chemical reactivity that allows them to interact readily with cellular constituents eliciting a plethora of biological activities. They are present exclusively in cruciferous vegetables, as glucosinolates, the intake of which has been associated with cancer chemoprevention. When the physical structure of these vegetables is disturbed, e.g. during mastication, the enzyme myrosinase is released and converts the glucosinolates to isothiocyanates (R-N=C=S), where R can be aliphatic or aromatic. Although sulforaphane, an aliphatic isothiocyanate, has received most attention worldwide, the most extensively studied aromatic isothiocyanate is phenethyl isothiocyanate (PEITC), and there are substantial differences in biological activity between the two sub-classes. In animal cancer models, PEITC effectively antagonized the carcinogenicity of chemicals, especially nitrosocompounds. A principal mechanism of their action is to protect the integrity of DNA by decreasing the levels of the genotoxic metabolites of chemical carcinogens. Extensive studies established that PEITC modulates the metabolism of the tobacco-specific carcinogenic nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by inhibiting its cytochrome P450-mediated bioactivation. Moreover, PEITC is a potent inducer of detoxification enzymes such as quinone reductase, glutathione S-transferase and glucuronosyl transferase. PEITC is rapidly absorbed and is characterized by a large bioavailability; Cmax concentrations achieved in plasma after dietary intake are sufficient to modulate carcinogen metabolism. PEITC is primarily metabolized by glutathione conjugation and is excreted in the urine and bile as the mercapturate. The ability of PEITC to perturb carcinogen metabolism through modulation of cytochrome P450 and phase II detoxification enzymes is comprehensively and critically reviewed.

Background: Phenethyl isothiocyanate (PEITC), the most comprehensively studied aromatic isothiocyanate, has been shown to act as an anti-cancer agent mainly through modulation of biotransformation enzymes responsible for metabolizing carcinogens in the human body. Humans are often exposed to carcinogenic factors, some of which through the diet, such as polycyclic aromatic hydrocarbon benzo[a]pyrene via the consumption of over-cooked meats. Inhibition of the enzymes responsible for the bioactivation of this carcinogen, for example CYP1A1, the major enzyme required for polycyclic aromatic hydrocarbons (PAHs) bioactivation, is recognized as a chemoprevention strategy. Objective: To evaluate the inhibitory effects of PEITC against benzo[a]pyreneinduced rise in rat liver CYP1A1 mRNA and apoprotein levels. Materials and Methods: Precision cut rat liver slices were treated with benzo[a]pyrene at 1 and 5 µM in the presence of PEITC (1-25 µM) for 24 hours, followed by determination of CYP1A1 mRNA and apoprotein levels using quantitative polymerase chain reaction and immunoblotting. Results: Findings revealed that PEITC inhibited benzo[a]pyrene-induced rise in rat liver CYP1A1 mRNA in a dose-dependent manner as well as the apoprotein levels of CYP1A. Conclusions: It was demonstrated that PEITC can directly inhibit the bioactivation of benzo[a]pyrene, indicating chemopreventive potential.

The principal objectives of our study were to ascertain whether sulforaphane, at dietary levels of intake, modulates rat hepatic cytochrome P450 and phase II enzyme systems and to evaluate the impact of such changes in the chemopreventive activity of this isothiocyanate. Animals were exposed to sulforaphane in their drinking water for 10 days, equivalent to daily doses of 3 and 12 mg/kg. Depentylation of pentoxyresorufin decreased and was paralleled by a decline in CYP2B apoprotein levels. At the higher dose, erythromycin N-demethylase activity declined and was accompanied by a similar decrease in CYP3A2 apoprotein levels. However, sulforaphane treatment upregulated CYP1A2 levels, determined immunologically, but the dealkylations of methoxy- and ethoxyresorufin were not similarly increased. Hepatic S9 preparations from sulforaphane-treated rats were less effective than control preparations in converting IQ (2-amino-3-methylimidazo-[4,5-f]quinoline) to mutagenic intermediates in the Ames test. To clarify the underlying mechanism, in vitro studies were undertaken. In β-naphthoflavone-treated rats, the inhibition by sulforaphane of the O-dealkylations of methoxy- and ethoxyresorufin was enhanced if the isothiocyanate was preincubated in the presence of NADPH. It may be inferred that sulforaphane induces hepatic CYP1A2 but the enzyme is not catalytically competent because of bound sulforaphane metabolite(s). Finally, sulforaphane stimulated, in a dose-dependent fashion, quinone reductase but failed to influence glutathione S-transferase, epoxide hydrolase and glucuronosyl transferase activities. It is concluded that, even at dietary doses, sulforaphane can modulate the xenobiotic-metabolising enzyme systems, shifting the balance of carcinogen metabolism toward deactivation, and this may be an important mechanism of its chemopreventive activity. © 2005 Wiley-Liss, Inc.

A reproducible method has been developed and validated that allows the detection of mutagenic material in human urine following the intake of a meal containing pan-fried beef patties. The mutagens are extracted from the urine with blue rayon and eluted with methanol/ammonia (100:1). Using 14C-2-amino-3-methylimidazo[4,5-f]quinoline (14C-IQ) as a tracer, the extraction efficiency of heterocyclic amines was consistently found to exceed 90%. It was observed that the critical factor in attaining high extraction efficiencies was the concentration of blue rayon, which depended on the nature of the matrix. Constituents of human urine were demonstrated to compete with IQ for the binding sites on the blue rayon. For the determination of the mutagenic activity in the extracted urine, the O-acetylase over-expressing Salmonella typhimurium strain YG1024 was utilised, and the Ames test procedure was optimised with respect to S9 concentration and incorporation of a pre-incubation step. Using the developed procedure, a marked rise in mutagenic activity, determined both in the absence and presence of an activation system, was evident in human urine following the intake of a beef patty meal. Normal mutagenicity levels were restored within 24 h. Finally, a method is described, once again employing blue rayon, for the extraction of heterocyclic amines from cooked beef patties with a 92% efficiency, as exemplified by IQ.

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 ability of cattle and deer liver to catalyse xenobiotic conjugation reactions was investigated and compared with that of the rat. Marked differences in the activity of these enzymes were noted between the domestic animals and rats. Hepatic microsomal epoxide hydrolase activity in cattle and deer, determined using benzo[a]pyrene 4,5-oxide as substrate, was nearly twice that of the rat. In contrast, glutathione S-transferase activity in hepatic cytosol, determined with 1-chloro-2,4-dinitrobenzene as substrate, was significantly lower in the cattle and deer. When 1,2-dichloro-4-nitrobenzene served as the accepting substrate, no activity was detectable in the cattle and deer. Similarly, glutathione reductase activity and total glutathione levels were markedly lower in the cattle and deer compared with the rat. Cytosolic sulfotransferase activity, monitored using 2-naphthol as substrate, was higher in cattle compared with the rat. Finally, microsomal UDP-glucuronosyl transferase activity, determined using 1-napththol as substrate, did not differ significantly among the three species.

1. The use of herbal products to treat a wide range of conditions is rising rapidly, leading to increased intake of phytochemicals. Recent studies revealed potentially fatal interactions between herbal remedies and traditional drugs. 2. In transplant patients, self-medication with St John's wort (Hypericum perforatum) has led to a drop in plasma levels of the immunosuppressant drug cyclosporine, causing tissue rejection. 3. Intake of St John's wort increases the expression of intestinal P-glycoprotein and the expression of CYP3A4 in the liver and intestine. The combined up-regulation in intestinal P-glycoprotein and hepatic and intestinal CYP3A4 impairs the absorption and stimulates the metabolism of cyclosporine, leading to subtherapeutic plasma levels. The St John's wort component, hyperforin, contributes to the induction of CYP3A4. 4. St John's wort also enhances the metabolism of other CYP3A4 substrates including the protease inhibitors indinavir and nevirapine, oral contraceptives, and tricyclic antidepressants such as amitriptyline. 5. Other herbal remedies with the potential to modulate cytochrome P450 activity and thus participate in interactions with conventional drugs include Milk thistle, Angelica dahurica, ginseng, garlic preparations, Danshen and liquorice. 6. Herbal products are currently not subject to the rigorous testing indispensable for conventional drugs. However, if potential drug interactions are to be predicted, it is essential that the ability of herbal products to interfere with drug-metabolizing enzyme systems is fully established.

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.

The present study was undertaken to investigate whether the consumption of green tea and black tea influences the excretion of mutagens and promutagens in rats treated orally with the food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Rats were maintained on aqueous extracts (2.5%, w/v) of green tea, black tea or decaffeinated black tea as their sole drinking liquid. After 4 weeks, the animals received, by gastric intubation, a single dose of IQ (5 mg/kg), and urine was collected for 48 h. Direct and indirect mutagenicity, in the presence of an activation system derived from Aroclor 1254-treated rats, was determined in the urine samples using the Ames mutagenicity assay. Consumption of green tea and black tea, but not of decaffeinated black tea, markedly decreased the urinary excretion of mutagens and promutagens. In a further study, supplementation of decaffeinated black tea with caffeine suppressed the excretion of mutagens and promutagens in the urine of rats pretreated with IQ. It is concluded that both green tea and black tea modulate the bioactivation and metabolism of IQ, and that caffeine is largely responsible for this effect.

1. Objectives were two-fold: (1) to compare the viability of precision-cut liver slices in two culture systems, namely the dynamic organ and the multiwell plate; and (2) to evaluate whether increasing the number of slices per incubation results in a proportional increase in the extent of metabolism. 2. With both culturing systems, the major products of 7-ethoxycoumarin metabolism were the sulphate and glucuronide conjugates of 7-hydroxycoumarin with very low levels of the free compound. When the multiwell plate procedure was used, metabolism increased linearly for at least 10 h, whereas it tended to plateau after 6 h in the dynamic organ culture system. At preincubations10 h, significantly more metabolism of 7-ethoxycoumarin was seen in the slices cultured using the multiwell system compared with the dynamic organ system. 3. Morphological evaluation employing light and electron microscopy revealed that liver slices incubated using the multiwell system were structurally better preserved compared with those incubated using the dynamic organ system. 4. Using the multiwell system, increasing the number of slices per incubation from one to two resulted in only a modest increase in the metabolism of 7-ethoxycoumarin. The rate of metabolism of this substrate was much higher with one liver slice when expressed per mg homogenate protein. 5. It is concluded that (1) the multiwell plate culture system for culturing slices is superior to the dynamic organ system in studying the metabolism of xenobiotics following long-term incubations, (2) increasing the number of slices per incubation does not result in a corresponding increase in the rate of metabolism, and (3) in both culture systems optimal viability appears to be within 24 h of incubation.

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.

There are two forms of diabetes, insulin-dependent Diabetes mellitus (IDDM) and non-insulin dependent Diabetes mellitus (NIDDM). There is evidence to suggest that reactive oxygen is involved in the pathogenicity and complications arising from IDDM, but there is little to suggest a role of oxidative stress in the pathogenesis of NIDDM. In order to investigate this hypothesis further, peripheral blood samples were taken from control individuals and IDDM and NIDDM patients and examined for antioxidant capacity and in the Comet assay for DNA strand breakage. The individuals answered a questionnaire to provide information relating to lifestyle factors in case such factors might have a confounding effect. There were 20 controls, 22 IDDM patients and 23 NIDDM patients. No differences could be detected in control and diabetic patient groups in terms of creatinine levels and antioxidant capacity. DNA damage in the Comet assay was at a lower level than in the control in the IDDM patients and a slightly lower level in the NIDDM patients, which might indicate that these cells are handling more oxidative damage on a regular basis. As expected, there were differences in glycosylated haemoglobin (HbA(1C)) levels. The confounding factors (smoking, drinking and vitamin intakes, etc.) appeared to have no effect.

The molecular dimensions and electronic structures of the first group of 100 US NCI/NTP miscellaneous chemicals, evaluated for potential carcinogenicity by computer-optimized molecular parametric analysis for chemical toxicity (COMPACT) have been re-determined. Using improved criteria for cytochrome P450 (CYP) substrate specificity, re-defined for CYP1 as having a COMPACT radius [square root of (deltaE - 9.5)2 + (a/d(2) - 7.8)2] of < 6.5, and for CYP2E as having a collision diameter of 6.5 angstroms or less and deltaE < 15.5, the likely substrates of CYP1 and CYP2E, which are regarded as potential carcinogens, have been identified. In addition, log P values have been taken into account; those chemicals with log P < 0 are non-lipophilic substrates unlikely to reach the activating cytochrome enzymes, and have been regarded as non-carcinogens. The second group of 100 US NCI/NTP chemicals have also now been categorized by COMPACT into CYP1 and CYP2E substrates, and their potential carcinogenicities evaluated. Of the 203 chemicals in the 2 groups, those positive in the rodent two-species life-span carcinogenicity study (rodent assay) were 53%, those positive in the Ames test (mutagenicity) were 48%, and those positive in the COMPACT programme (carcinogenicity, mutagenicity, cytotoxicity) were 54%. Concordance between the COMPACT prediction of carcinogenicity/cytotoxicity and rodent two species life-span carcinogenicity data for the 203 chemicals is 69%, and correlation of COMPACT with Ames test data is 61%. The sensitivity of COMPACT for predicting rodent carcinogenicity is 72%, whereas the sensitivity of the Ames test for predicting carcinogenicity for the 203 chemicals was only 57%. The degree (severity) of rodent carcinogenicity also showed correlation with the COMPACT predictive evaluations of the chemicals.