Neuroprotective Effects of 7-Geranyloxycinnamic Acid from Melicope lunu ankenda Leaves.

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.

Induction of Apoptosis by Gluconasturtiin-Isothiocyanate (GNST-ITC) in Human Hepatocarcinoma HepG2 Cells and Human Breast Adenocarcinoma MCF-7 Cells.

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 µM and 5.02 µM, 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.

Induction of Apoptosis and Cytotoxicity by Raphasatin in Human Breast Adenocarcinoma MCF-7 Cells.

Glucoraphasatin (GRH), a glucosinolate present abundantly in the plants of the Brassicaceae family, is hydrolyzed by myrosinase to raphasatin, which is considered responsible for its cancer chemopreventive activity; however, the underlying mechanisms of action have not been investigated, particularly in human cell lines. The aims of this study are to determine the cytotoxicity of raphasatin, and to evaluate its potential to cause apoptosis and modulate cell cycle arrest in human breast adenocarcinoma MCF-7 cells. The cytotoxicity was determined following incubation of the cells with glucoraphasatin or raphasatin (0⁻100 µM), for 24, 48, and 72 h. GRH displayed no cytotoxicity as exemplified by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. When myrosinase was added to the incubation system to convert GRH to raphasatin, cytotoxicity was evident. Exposure of the cells to raphasatin stimulated apoptosis, as was exemplified by cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation. Moreover, using Annexin V-FITC assay, raphasatin induced apoptosis, as witnessed by changes in cellular distribution of cells, at different stages of apoptosis; in addition, raphasatin caused the arrest of the MCF-7 cells at the G2 + M phase. In conclusion, raphasatin demonstrated cancer chemopreventive potential against human breast adenocarcinoma (MCF-7) cells, through induction of apoptosis and cell cycle arrest.

Synergistic and antagonistic interactions of binary mixtures of polycyclic aromatic hydrocarbons in the upregulation of CYP1 activity and mRNA levels in precision-cut rat liver 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.

A principal mechanism for the cancer chemopreventive activity of phenethyl isothiocyanate is modulation of carcinogen metabolism.

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.

A glucosinolate-rich extract of Japanese Daikon perturbs carcinogen-metabolizing enzyme systems in rat, being a potent inducer of hepatic glutathione S-transferase.

PURPOSE: Glucosinolates/isothiocyanates are an established class of naturally occurring chemopreventive agents, a principal mechanism of action being to limit the generation of genotoxic metabolites of chemical carcinogens, as a result of modulation of cytochrome P450 and phase II detoxification enzymes. The objective of this study was to assess whether a glucosinolate-rich extract from Daikon sprouts, containing glucroraphasatin and glucoraphenin, is a potential chemopreventive agent by modulating such enzymes in the liver and lung of rats. METHODS: Rats were exposed to the glucosinolate-rich Daikon extract through the diet, at three dose levels, for 14 days, so that the low dose simulates dietary intake. RESULTS: At the low dose only, a modest increase was noted in the hepatic dealkylations of methoxy-, ethoxy-, pentoxyresorufin and benzyloxyquinoline that was accompanied by elevated expression of CYP1 and CYP3A2 apoprotein levels. In lung, only a modest increase in the dealkylation of pentoxyresorufin was observed. At higher doses, in both tissues, these increases were abolished. At the same low dietary dose, the Daikon extract elevated markedly glutathione S-transferase activity paralleled by rises in GSTα, GSTµ and GSTπ protein expression. An increase was also noted in quinone reductase activity and expression. Finally, glucuronosyl transferase and epoxide hydrolase activities and expression were also up-regulated, but necessitated higher doses. CONCLUSION: Considering the ability of Daikon glucosinolates to effectively enhance detoxification enzymes, in particular glutathione S-transferase, it may be inferred that consumption of this vegetable may possess significant chemopreventive activity and warrants further evaluation through epidemiology and studies in animal models of cancer.

Up-regulation of cytochrome P450 and phase II enzymes by xenobiotics in precision-cut tissue slices.

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.

Debrisoquine metabolism and CYP2D expression in marmoset liver microsomes.

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.

4-Methylsulfanyl-3-butenyl isothiocyanate derived from glucoraphasatin is a potent inducer of rat hepatic phase II enzymes and a potential chemopreventive agent.

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.

The naturally occurring aliphatic isothiocyanates sulforaphane and erucin are weak agonists but potent non-competitive antagonists of the aryl hydrocarbon receptor.

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.

The natural chemopreventive phytochemical R-sulforaphane is a far more potent inducer of the carcinogen-detoxifying enzyme systems in rat liver and lung than the S-isomer.

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.

Exposure to isothiocyanates suppresses urinary mutagenicity in rats treated with heterocyclic amine IQ: lack of association with CYP1 activity.

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.

Induction of epoxide hydrolase and glucuronosyl transferase by isothiocyanates and intact glucosinolates in precision-cut rat liver slices: importance of side-chain substituent and chirality.

The potential of three isothiocyanates, namely R,S-sulforaphane, erucin and phenethyl isothiocyanate, of two naturally occurring glucosinolates, namely glucoerucin and glucoraphanin, and of the enantiomers of sulforaphane to modulate glucuronosyl transferase and epoxide hydrolase, two major carcinogen-metabolising enzyme systems, was investigated in precision-cut rat liver slices. Following exposure of the slices to the isothiocyanates (0-25 µM), erucin and phenethyl isothiocyanate, but not R,S-sulforaphane, elevated glucuronosyl transferase and epoxide hydrolase activities and expression, determined immunologically. Of the two enantiomers of sulforaphane, the R-enantiomer enhanced, whereas the S-enantiomer impaired, glucuronosyl transferase activity and only the former increased protein expression; furthermore, R-sulforaphane was more effective than the S-enantiomer in up-regulating microsomal epoxide hydrolase. When precision-cut rat liver slices were exposed to the same concentrations of glucoerucin and glucoraphanin, both glucosinolates caused a marked increase in the activity and expression of the microsomal epoxide hydrolase but had no effect on glucuronosyl transferase activity. It may be inferred that the ability of isothiocyanates to enhance hepatic microsomal epoxide hydrolase and glucuronosyl transferase activities is dependent on the nature of the side chain. Moreover, in the case of sulforaphane, the naturally occurring R-enantiomer increased both activities, whereas, in contrast, activities were impaired in the case of the S-enantiomer. Finally, intact glucosinolates are potent inducers of epoxide hydrolase and can thus contribute directly to the chemopreventive potential associated with cruciferous vegetable consumption.

Phenethyl isocyanate is not the metabolite of phenethyl isothiocyanate responsible for mechanism-based inhibition of cytochrome P450.

Phenethyl isothiocyanate is a chemopreventive phytochemical present in cruciferous vegetables where it exists as the glucosinolate gluconasturtiin. It is a mechanism-based inhibitor of both rat and human cytochrome P450 enzymes. The principal objective of the present study was to ascertain whether phenethyl isocyanate, formed by the cytochrome P450-mediated oxidative desulphuration of phenethyl isothiocyanate, is the metabolite responsible for the mechanism-based inhibition. Phenethyl isothiocyanate, following incubation with Aroclor 1254-induced rat liver microsomes in the presence of NADPH, markedly suppressed the CYP1A-mediated O-deethylation of ethoxyresorufin; extent of inhibition was directly related to the pre-incubation time and was antagonised by reduced glutathione. When human liver microsomes were used, the inhibitory effect of phenethyl isothiocyanate, which was once again related to the pre-incubation time, was even more pronounced. When the ability of phenethyl isothiocyanate and phenethyl isocyanate to directly inhibit the O-deethylation of ethoxyresorufin in rat microsomes was compared, the latter compound was only moderately more effective. In human microsomes, both compounds were equipotent. In phenobarbital-induced lung microsomes, phenethyl isothiocyanate was a direct and potent inhibitor of the O-depentylation of pentoxyresorufin; pre-incubation of the isothiocyanate had no impact. Human precision-cut liver slices were more effective than rat slices in metabolising phenethyl isothiocyanate. Pre-treatment of rats, however, with phenobarbitone significantly enhanced the metabolism of isothiocyanate. It may be inferred from the present studies that: (a) phenethyl isocyanate is not the metabolite of phenethyl isothiocyanate responsible for its mechanism-based inhibition, and (b) CYP2B is an important catalyst of the metabolism of phenethyl isothiocyanate.

Effect of 2-amino-9H-pyrido[2,3-b]indole (AalphaC), a carcinogenic heterocyclic amine present in food, on atherosclerotic plaque development in apoE deficient mice.

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.

Up-regulation of the glutathione S-transferase system in human liver by polycyclic aromatic hydrocarbons; comparison with rat liver and lung.

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.

Interactions between polycyclic aromatic hydrocarbons in binary mixtures: effects on gene expression and DNA adduct formation in precision-cut rat liver slices.

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.

Up-regulation of the CYP1 family in rat and human liver by the aliphatic isothiocyanates erucin and sulforaphane.

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.

Differential response of human and rat epoxide hydrolase to polycyclic aromatic hydrocarbon exposure: studies using precision-cut tissue slices.

The potential of polycyclic aromatic hydrocarbons (PAHs) to modulate microsomal epoxide hydrolase activity, determined using benzo[a]pyrene 5-oxide as substrate, in human liver, was evaluated and compared to rat liver. Precision-cut liver slices prepared from fresh human liver were incubated with six structurally diverse PAHs, at a range of concentrations, for 24h. Of the six PAHs studied, benzo[a]pyrene, dibenzo[a,h]anthracene and fluoranthene gave rise to a statistically significant increase in epoxide hydrolase activity, which was accompanied by a concomitant increase in epoxide hydrolase protein levels determined by immunoblotting. The other PAHs studied, namely dibenzo[a,l]pyrene, benzo[b]fluoranthene and 1-methylphenanthrene, influenced neither activity nor enzyme protein levels. When rat slices were incubated under identical conditions, only benzo[a]pyrene and dibenzo[a,h]anthracene elevated epoxide hydrolase activity, which was, once again accompanied by a rise in protein levels. At the mRNA level, however, all six PAHs caused an increase, albeit to different extent. In rat, epoxide hydroxylase activity in lung slices was much lower than in liver slices. In lung slices, epoxide hydrolase activity was elevated following exposure to benzo[a]pyrene and dibenzo[a,l]pyrene and, to a lesser extent, 1-methylphenanthrene; similar observations were made at the protein level. At both activity and protein levels extent of induction was far more pronounced in the lung compared with the liver. It is concluded that epoxide hydrolase activity is an inducible enzyme by PAHs, in both human and rat liver, but induction potential by individual PAHs varies enormously, depending on the nature of the compound involved. Marked tissue differences in the nature of PAHs stimulating activity in rat lung and liver were noted. Although in the rat basal lung epoxide hydrolase activity is much lower than liver, it is more markedly inducible by PAHs.

Up-regulation of CYP1A/B in rat lung and liver, and human liver precision-cut slices by a series of polycyclic aromatic hydrocarbons; association with the Ah locus and importance of molecular size.

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.