Gene expression profile of primary prostate epithelial and stromal cells in response to sulforaphane or iberin exposure

BACKGROUND: Broccoli consumption has been associated with a reduced risk of prostate cancer. Isothiocyanates (ITCs) derived from glucosinolates that accumulate in broccoli are dietary compounds that may mediate these health effects. Sulforaphane (SF, 4-methylsulphinylbutyl ITC) derives from heading broccoli (calabrese) and iberin (IB, 3-methylsulphinypropyl ITC) from sprouting broccoli. While there are many studies regarding the biological activity of SF, mainly undertaken with cancerous cells, there are few studies associated with IB. METHODS: Primary epithelial and stromal cells were derived from benign prostatic hyperplasia tissue. Affymetrix U133 Plus 2.0 whole genome arrays were used to compare global gene expression between these cells, and to quantify changes in gene expression following exposure to physiologically appropriate concentrations of SF and IB. Ontology and pathway analyses were used to interpret results. Changes in expression of a subset of genes were confirmed by real-time RT-PCR. RESULTS: Global gene expression profiling identified epithelial and stromal-specific gene expression profiles. SF induced more changes in epithelial cells, whereas IB was more effective in stromal cells. Although IB and SF induced different changes in gene expression in both epithelial and stromal cells, these were associated with similar pathways, such as cell cycle and detoxification. Both ITCs increased expression of PLAGL1, a tumor suppressor gene, in stromal cells and suppressed expression of the putative tumor promoting genes IFITM1, CSPG2, and VIM in epithelial cells. CONCLUSION: These data suggest that IB and SF both alter genes associated with cancer prevention, and IB should be investigated further as a potential chemopreventative agent.

The GSTM1 null genotype confers an increased risk for Solar Keratosis Development in an Australian Caucasian Population

Solar keratoses affect approximately 50% of Australian Caucasians aged over 40 y. Solar keratoses can undergo malignant transformation into squamous cell carcinoma followed by possible metastasis and are risk factors for basal cell carcinoma, melanoma, and squamous cell carcinoma. The glutathione-S-transferase genes play a part in detoxification of carcinogens and mutagens, including some produced by ultraviolet radiation. This study examined the role of glutathione-S-transferase M1, T1, P1, and Z1 gene polymorphisms in susceptibility to solar keratoses development. Using DNA samples from volunteers involved in the Nambour Skin Cancer Prevention Trial, allele and genotype frequencies were determined using polymerase chain reaction and restriction enzyme digestion. No significant differences were detected in glutathione-S-transferase P1 and glutathione-S-transferase Z1 allele or genotype frequencies; however, a significant association between glutathione-S-transferase M1 genotypes and solar keratoses development was detected (p=0.003) with null individuals having an approximate 2-fold increase in risk for solar keratoses development (odds ratio: 2.1; confidence interval: 1.3-3.5) and a significantly higher increase in risk in conjunction with high outdoor exposure (odds ratio: 3.4; confidence interval: 1.9-6.3). Also, a difference in glutathione-S-transferase T1 genotype frequencies was detected (p=0.039), although considering that multiple testing was undertaken, this was found not to be significant. Fair skin and inability to tan were found to be highly significant risk factors for solar keratoses development with odds ratios of 18.5 (confidence interval: 5.7-59.9) and 7.4 (confidence interval: 2.6-21.0), respectively. Overall, glutathione-S-transferase M1 conferred a significant increase in risk of solar keratoses development, particularly in the presence of high outdoor exposure and synergistically with known phenotypic risk factors of fair skin and inability to tan.

The null allele of GSTM1 does not affect susceptibility to solar keratoses in the Australian white population

Solar keratoses (SKs) are induced by exposure to UV radiation and are capable of undergoing transformation to squamous cell carcinoma (SCC).1 The two main factors influencing the occurrence of SK are the sensitivity of the skin to sunlight and the total duration of solar exposure. These factors are responsible for the high incidence of SK in Australia. Although the influence of genetic factors is not defined, there is evidence that the gene encoding the enzyme, glutathione S-transferase, may be implicated in cancer predisposition and therefore SK. Glutathione S-transferase Mu-1 (GSTM1) is an isoenzyme involved in the detoxification of carcinogens. The GSTM1 protein is completely absent in approximately 50% of white persons. This absence is caused by a homozygous gene deletion on chromosome 1p resulting in a null genotype.2 Katoh3 showed that the frequency of the GSTM1 null genotype was significantly higher in 85 patients with urothelial cancer (61.2%; p < 0.05), suggesting that the null genotype may increase cancer susceptibility. This finding was supported by Lafuente et al.4 who found evidence that persons who lack the GSTM1 gene have approximately twice the chance of experiencing malignant melanoma. Further research in the United Kingdom found that patients with two or more skin tumors of different types, basal cell carcinoma (BCC) and SCC, had a significantly higher frequency of GSTM1 null genotypes than controls (71%; p = 0.033). However the GSTM1 genotype in patients with only SCC was not excessive in this population.5 Persons residing in northern Australia have the highest incidence of nonmelanoma skin cancer (SCC and BCC) in the world6 and receive far greater solar exposure than persons residing in the United Kingdom. It is possible that the GSTM1 null genotype may affect susceptibility to SK, which may act as SCC precursors, in Australians exposed to these high levels of solar radiation.

Adaptations to a subterranean environment and longevity revealed by the analysis of mole rat genomes

Subterranean mammals spend their lives in dark, unventilated environments that are rich in carbon dioxide and ammonia and low in oxygen. Many of these animals are also long-lived and exhibit reduced aging-associated diseases, such as neurodegenerative disorders and cancer. We sequenced the genome of the Damaraland mole rat (DMR, Fukomys damarensis) and improved the genome assembly of the naked mole rat (NMR, Heterocephalus glaber). Comparative genome analyses, along with the transcriptomes of related subterranean rodents, revealed candidate molecular adaptations for subterranean life and longevity, including a divergent insulin peptide, expression of oxygen-carrying globins in the brain, prevention of high CO2-induced pain perception, and enhanced ammonia detoxification. Juxtaposition of the genomes of DMR and other more conventional animals with the genome of NMR revealed several truly exceptional NMR features: unusual thermogenesis, an aberrant melatonin system, pain insensitivity, and unique processing of 28S rRNA. Together, these genomes and transcriptomes extend our understanding of subterranean adaptations, stress resistance, and longevity.

Relevance of the deletion polymorphisms of the glutathione S-transferases GSTT1 and GSTM1 in pharmacology and toxicology

Although cytosolic glutathione S-transferase (GST) enzymes occupy a key position in biological detoxification processes, two of the most relevant human isoenzymes, GSTT1-1 and GSTM1-1, are genetically deleted (non-functional alleles GSTT1*0 and GSTM1*0) in a high percentage of the human population, with major ethnic differences. The structures of the GSTT and GSTM gene areas explain the underlying genetic processes. GSTT1-1 is highly conserved during evolution and plays a major role in phase-II biotransformation of a number of drugs and industrial chemicals, e.g. cytostatic drugs, hydrocarbons and halogenated hydrocarbons. GSTM1-1 is particularly relevant in the deactivation of carcinogenic intermediates of polycyclic aromatic hydrocarbons. Several lines of evidence suggest that hGSTT1-1 and/or hGSTM1-1 play a role in the deactivation of reactive oxygen species that are likely to be involved in cellular processes of inflammation, ageing and degenerative diseases. There is cumulating evidence that combinations of the GSTM1*0 state with other genetic traits affecting the metabolism of carcinogens (CYP1A1, GSTP1) may predispose the aero-digestive tract and lung, especially in smokers, to a higher risk of cancer. The GSTM1*0 status appears also associated with a modest increase in the risk of bladder cancer, consistent with a GSTM1 interaction with carcinogenic tobacco smoke constituents. Both human GST deletions, although largely counterbalanced by overlapping substrate affinities within the GST superfamily, have consequences when the organism comes into contact with distinct man-made chemicals. This appears relevant in industrial toxicology and in drug metabolism.

Enhancement of the chemoprotective enzymes glucuronosyl transferase and glutathione transferase in specific organs of the rat by the coffee components kahweol and cafestol

The coffee components kahweol and cafestol (K/C) have been reported to protect the colon and other organs of the rat against the formation of DNA adducts by 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) and aflatoxin B1. PhIP is a cooked-food mutagen to which significant human exposure and a role in colon cancer etiology are attributed, and, interestingly, such cancers appear to develop at a lower rate in consumers of coffees with high amounts of K/C. Earlier studies in rodent liver have shown that a key role in the chemopreventive effect of K/C is likely to be due to the potential of these compounds to induce the detoxification of xenobiotics by glutathione transferase (GST) and to enhance the synthesis of the corresponding co-factor glutathione. However, mutagens like PhIP may also be detoxified by UDP-glucuronosyl transferase (UDPGT) for which data are lacking regarding a potential effect of K/C. Therefore, in the present study, we investigated the effect of K/C on UDPGT and, concomitantly, we studied overall GST and the pattern of individual GST classes, particularly GST-θ, which was not included in earlier experiments. In addition, we analyzed the organ-dependence of these potentially chemopreventive effects. K/C was fed to male F344 rats at 0.122% in the chow for 10 days. Enzyme activities in liver, kidney, lung, colon, salivary gland, pancreas, testis, heart and spleen were quantified using five characteristic substrates and the hepatic protein pattern of GST classes α, μ, and π was studied with affnity chromatography/HPLC. Our study showed that K/C is not only capable of increasing overall GST and GST classes α, μ, and π but also of enhancing UDGPT and GST-θ. All investigated K/C effects were strongest in liver and kidney, and some response was seen in lung and colon but none in the other organs. In summary, our results show that K/C treatment leads to a wide spectrum of increases in phase II detoxification enzymes. Notably, these effects occurred preferentially in the well perfused organs liver and kidney, which may thus not only contribute to local protection but also to anti-carcinogenesis in distant, less stimulated organs such as the colon.

Rapid analysis of GSTM1, GSTT1 and GSTP1 polymorphisms using real-time polymerase chain reaction

Polymorphisms of glutathione transferases (GST) are important genetic determinants of susceptibility to environmental carcinogens (Rebbeck, 1997). The GSTs are a multigene family of dimeric enzymes involved in detoxification, and, in a few cases, the bioactivation of a variety of xenobiotics (Hayes et al., 1995). The cytosolic GST enzyme family consists of four major classes of enzymes, referred to as alpha, mu, pi and theta. Several members of this family (for example, GSTM1, GSTT1 and GSTP1) are polymorphic in human populations (Wormhoudt et al., 1999). Molecular epidemiology studies have examined the role of GST polymorphisms as susceptibility factors for environmentally and/or occupationally induced cancers (Wormhoudt et al., 1999). In particular, case-control studies showed a relationship between the GSTM1 null genotype and the development of cancer in association with smoking habits, which has been shown for cancers of the respiratory and gastrointestinal tracts as well as other cancer types (Miller et al., 1997). Only a few molecular epidemiological studies addressed the role of GSTT1 and GSTP1 polymorphisms in cancer susceptibility. Since GSTP1 is a key player in biotransformation/bioactivation of benzo(a)pyrene, GSTP1 may be even more important than GSTM1 in the prevention of tobacco-induced cancers (Harries et al., 1997; Harris et al., 1998). To date, this relationship has not been sufficiently addressed in humans. Comprehensive molecular epidemiological studies may add to the current knowledge of the role of GST polymorphisms in cancer susceptibility and extent of the knowledge gained from approaches that used phenotyping, such as GSTM1 activity as it relates to trans-stilbene oxide, or polymerase chain reaction (PCR) based genotyping of polymorphic isoenzymes (Bell et al., 1993; Pemble et al., 1994; Harries et al., 1997).

Conjugation of carcinogens by 6 class glutathione S-transferases : mechanisms and relevance to variations in human risk

Conjugation of chemicals with glutathione (GSH) can lead to decreased or increased toxicity. A genetic deficiency in the GSH S-transferase μ class gene M1 has been hypothesized to lead to greater risk of lung cancer in smokers. Recently a gene deletion polymorphism involving the human θ enzyme T1 has been described; the enzyme is present in erythrocytes and can be readily assayed. A rat θ class enzyme, 5-5, has structural and catalytic similarity and the protein was expressed in the Salmonella typhimurium tester strain TA1535. Expression of the cDNA vector increased the mutagenicity of ethylene dibromide and several methylene dihalides. Mutations resulting from the known GSH S-transferase substrate 1,2-epoxy-3-(4′nitrophenoxy)propane were decreased in the presence of the transferase. Expression of transferase 5-5 increased mutations when 1,2,3,4-diepoxybutane (butadiene diepoxide), 4-bromo-1,2-epoxybutane, or 1,3-dichloracetone were added. The latter compound is a model for the putative 1,2-dibromo-3-chloropropane oxidation product 1-bromo-3-chloroacetone. These genotoxicity and genotyping assays may be of use in further studies of the roles of GSH S-transferase θ enzymes in bioactivation and detoxication and any changes in risk due to polymorphism.

Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II)

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(II) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1 μM of complexed Hg(II), and for inhibition of motility it was 0.05 μM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 μM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned.

Influence of polymorphisms of the human glutathione transferases and cytochrome P450 2E1 enzyme on the metabolism and toxicity of ethylene oxide and acrylonitrile

A cohort of 59 persons with industrial handling of low levels of acrylonitrile is being studied as part of a medical surveillance programme. Previously, an extended haemoglobin adduct monitoring (N-(cyanoethyl)valine and N-(hydroxyethyl)-valine) was performed regarding the glutathione transferases hGSTM1 and hGSTT1 polymorphisms but no influence of hGSTM1 or hGSTT1 polymorphisms on specific adduct levels was found. A compilation of case reports of human accidental poisonings had pointed to significant individual differences in human acrylonitrile metabolism and toxicity. Therefore, a re-evaluation of the industrial cohort included known polymorphisms of the glutathione transferases hGSTM3 and hGSTP1 as well as of the cytochrome P450 CYP2E1. A detailed statistical analysis revealed that exposed carriers of the allelic variants of hGSTP1, hGSTP1*B/hGSTP1*C, characterized by a single nucleotide polymorphism at nucleotide 313 which results in a change from Ile to Val at codon 104, had higher levels of the acrylonitrile-specific haemoglobin adduct N-(cyanoethyl)valine compared to the carriers of the codon 113 alleles hGSTP1*A and hGSTP1*D. The single nucleotide polymorphism at codon 113 of hGSTP1 (hGSTP1*A/hGSTP1*B versus hGSTP1*C/hGSTP1*D) did not show an effect, and also no influence was seen on specific haemoglobin adduct levels of the polymorphisms of hGSTM3 or CYP2E1. The data, therefore, point to a possible influence of a human enzyme polymorphism of the GSTP1 gene at codon 104 on the detoxication of acrylonitrile which calls for experimental toxicological investigation. The study also confirmed the impact of GSTT1 polymorphism on background N-(hydroxyethyl)-valine adduct levels in haemoglobin which are caused by endogenous ethylene oxide.

Species differences in acrylonitrile metabolism and toxicity between experimental animals and humans based on observations in human accidental poisonings

The high acute toxicity of acrylonitrile may be a result of its intrinsic biological reactivity or of its metabolite cyanide. Intravenous N-acetylcysteine has been recommended for treatment of accidental intoxications in acrylonitrile workers, but such recommendations vary internationally. Acrylonitrile is metabolized in humans and experimental animals via two competing pathways; the glutathione-dependent pathway is considered to represent an avenue of detoxication whilst the oxidative pathway leads to a genotoxic epoxide, cyanoethylene oxide, and to elimination of cyanide. Cases of acute acrylonitrile overexposure or intoxication have occurred within persons having industrial contact with acrylonitrile; the route of exposure was by inhalation and/or by skin contact. The combined observations lead to the conclusion of a much higher impact of the oxidative metabolism of acrylonitrile in humans than in rodents. This is confirmed by differences in the clinical picture of acute life-threatening intoxications in both species, as well as by differential efficacies of antidotes. A combination of N-acetylcysteine with sodium thiosulfate seems an appropriate measure for antidote therapy of acute acrylonitrile intoxications. Clinical observations also highlight the practical importance of human individual susceptibility differences. Furthermore, differential adduct monitoring, assessing protein adducts with different rates of decay, enables the development of more elaborated biological monitoring strategies for the surveillance of workers with potential acrylonitrile contact.

Haemoglobin adducts of acrylonitrile and ethylene oxide in acrylonitrile workers, dependent on polymorphisms of the glutathione transferases GSTT1 and GSTM1

Fifty-nine persons with industrial handling of low levels of acrylonitrile (AN) were studied. As part of a medical surveillance programme an extended haemoglobin adduct monitoring [N-(cyanoethyl)valine, CEV; N- (methyl)valine, MV; N-(hydroxyethyl)valine, HEV] was performed. Moreover, the genetic states of the polymorphic glutathione transferases GSTM1 and GSTT1 were assayed by polymerase chain reaction (PCR). Repetitive analyses of CEV and MV in subsequent years resulted in comparable values (means, 59.8 and 70.3 μg CEV/1 blood; 6.7 and 6.7 μg MV/1 blood). Hence, the industrial AN exposures were well below current official standards. Monitoring the haemoglobin adduct CEV appears as a suitable means of biomonitoring and medical surveillance under such exposure conditions. There was also no apparent correlation between the CEV and HEV or CEV and MV adduct levels. The MV and HEV values observed represented background levels, which apparently are not related to any occupational chemical exposure. There was no consistent effect of the genetic GSTM1 or GSTT1 state on CEV adduct levels induced by acrylonitrile exposure. Therefore, neither GSTM1 nor GSTT1 appears as a major AN metabolizing isoenzyme in humans. The low and physiological background levels of MV were also not influenced by the genetic GSTM1 state, but the MV adduct levels tended to be higher in GSTT1- individuals compared to GSTT1 + persons. With respect to the background levels of HEV adducts observed, there was no major influence of the GSTM1 state, but GST- individuals displayed adduct levels that were about 1/3 higher than those of GSTT1+ individuals. The coincidence with known differences in rates of background sister chromatid exchange between GSTT1- and GSTT1 + persons suggests that the lower ethylene oxide (EO) detoxification rate in GSTT1- persons, indicated by elevated blood protein hydroxyethyl adduct levels, leads to an increased genotoxic effect of the physiological EO background.

Possible impact of human CYP2E1 polymorphisms on the metabolism of acrylonitrile

Case reports of human accidental poisonings point to significant individual differences in human acrylonitrile metabolism and toxicity. A cohort of 59 persons with industrial handling of low levels of acrylonitrile has repetitively been studied from 1994 through 1999 as part of a medical surveillance programme. The analyses included adduct determinations of N-terminal N-(cyanoethyl)valine in haemoglobin and genotypings of the following cytochrome P-450 2E1 (CYP2E1) polymorphisms: G-1259C and C-1019T (two subjects heterozygous), A-316G (three subjects heterozygous), T-297A (15 subjects heterozygous), G-35T (eight subjects heterozygous), G4804A (two subjects heterozygous), T7668A (six subjects heterozygous). N-(Cyanoethyl)valine adduct levels were, if any, only slightly influenced by smoking and mainly determined by the external acrylonitrile exposures. The individual means and medians of N-(cyanoethyl)valine levels over the entire observation period were compared with the CYP2E1 variants (Wilcoxon rank sum test). No influences of the investigated CYP2E1 polymorphisms on the N-(cyanoethyl)valine levels appeared at the 5% level. However, there was a trend, at a level of P≃0.1, pointing to higher acrylonitrile-specific adduct levels in persons with the A-316G mutation. Higher adduct levels would be compatible with a slower CYP2E1-mediated metabolism of acrylonitrile and with lower extents of toxification to cyanide.

Substance use disorders

Substance use disorders involve alcohol and a range of other legal and illicit drugs, and are characterised by a preoccupation with or craving for the substance, a greater priority to substance use than other goals, and/or a difficulty controlling consumption. Use of the substance may continue despite negative impacts on other activities, roles, relationships, and physical and mental health. Increased physical tolerance to the substance and withdrawal symptoms may also occur. Broad impacts on social and cognitive functioning and on physical and mental health emerge with increasing problem severity. Diffuse cognitive impairment may persist for up to 12 months post-detoxification in alcohol dependence. Psychological comorbidity is common, particularly mood and anxiety disorders. A quarter of all Australians will have a substance use disorder in their lifetime. One in five will consume alcohol at a level that puts them at risk of harm from an alcohol-related disease or injury over their lifetime. Australians aged 18 to 29 years are at higher risk than other age groups.

Stereochemical and steric control of enzymatic glucuronidation : A rational approach for the design of novel Inhibitors for the human UDP-glucuronosyltransferase 2B7

The UDP-glucuronosyltransferases (UGTs) are enzymes of the phase II metabolic system. These enzymes catalyze the transfer of α-D-glucuronic acid from UDP-glucuronic acid to aglycones bearing nucleophilic groups affording exclusively their corresponding β-D-glucuronides to render lipophilic endobiotics and xenobiotics more water soluble. This detoxification pathway aids in the urinary and biliary excretion of lipophilic compounds thus preventing their accumulation to harmful levels. The aim of this study was to investigate the effect of stereochemical and steric features of substrates on the glucuronidation catalyzed by UGTs 2B7 and 2B17. Furthermore, this study relates to the design and synthesis of novel, selective inhibitors that display high affinity for the key enzyme involved in drug glucuronidation, UGT2B7. The starting point for the development of inhibitors was to assess the influence of the stereochemistry of substrates on the UGT-catalyzed glucuronidation reaction. A set of 28 enantiomerically pure alcohols was subjected to glucuronidation assays employing the human UGT isoforms 2B7 and 2B17. Both UGT enzymes displayed high stereoselectivity, favoring the glucuronidation of the (R)-enantiomers over their respective mirror-image compounds. The spatial arrangement of the hydroxy group of the substrate determined the rate of the UGT-catalyzed reaction. However, the affinity of the enantiomeric substrates to the enzymes was not significantly influenced by the spatial orientation of the nucleophilic hydroxy group. Based on these results, a rational approach for the design of inhibitors was developed by addressing the stereochemical features of substrate molecules. Further studies showed that the rate of the enzymatic glucuronidation of substrates was also highly dependent on the steric demand in vicinity of the nucleophilic hydroxy group. These findings provided a rational approach to turn high-affinity substrates into true UGT inhibitors by addressing stereochemical and steric features of substrate molecules. The tricyclic sesquiterpenols longifolol and isolongifolol were identified as high-affinity substrates which displayed high selectivity for the UGT isoform 2B7. These compounds served therefore as lead structures for the design of potent and selective inhibitors for UGT2B7. Selective and potent inhibitors were prepared by synthetically modifying the lead compounds longifolol and isolongifolol taking stereochemical and steric features into account. The best inhibitor of UGT2B7, β-phenyllongifolol, displayed an inhibition constant of 0.91 nM.