Ward G. Kirlin

Glutathione redox potential in response to differentiation and enzyme inducers

The reduced glutathione (GSH)/oxidized glutathione (GSSG) redox state is thought to function in signaling of detoxification gene expression, but also appears to be tightly regulated in cells under normal conditions. Thus it is not clear that the magnitude of change in response to physiologic stimuli is sufficient for a role in redox signaling under nontoxicologic conditions. The purpose of this study was to determine the change in 2GSH/GSSG redox during signaling of differentiation and increased detoxification enzyme activity in HT29 cells. We measured GSH, GSSG, cell volume, and cell pH, and we used the Nernst equation to determine the changes in redox potential Eh of the 2GSH/GSSG pool in response to the differentiating agent, sodium butyrate, and the detoxification enzyme inducer, benzyl isothiocyanate. Sodium butyrate caused a 60-mV oxidation (from -260 to -200 mV), an oxidation sufficient for a 100-fold change in protein dithiols:disulfide ratio. Benzyl isothiocyanate caused a 16-mV oxidation in control cells but a 40-mV oxidation (to -160 mV) in differentiated cells. Changes in GSH and mRNA for glutamate:cysteine ligase did not correlate with Eh; however, correlations were seen between Eh and glutathione S-transferase (GST) and nicotinamide adenine dinucleotide phosphate (NADPH):quinone reductase activities (N:QR). These results show that 2GSH/GSSG redox changes in response to physiologic stimuli such as differentiation and enzyme inducers are of a sufficient magnitude to control the activity of redox-sensitive proteins. This suggests that physiologic modulation of the 2GSH/GSSG redox poise could provide a fundamental parameter for the control of cell phenotype.

Cysteine/cystine couple is a newly recognized node in the circuitry for biologic redox signaling and control

Redox mechanisms function in control of gene expression, cell proliferation, and apoptosis, but the circuitry for redox signaling remains unclear. Cysteine and methionine are the only amino acids in proteins that undergo reversible oxidation/reduction under biologic conditions and, as such, provide a means for control of protein activity, protein‐protein interaction, protein trafficking, and protein‐DNA interaction. Hydrogen peroxide and other reactive oxygen species (ROS) provide a mechanism to oxidize signaling proteins. However, oxidation of sulfur‐ containing side chains of cysteine and methionine by ROS can result in oxidation states of sulfur (e.g., sulfinate, sulfonate, sulfone) that are not reducible under biologic conditions. Thus, mechanisms for oxidation that protect against over‐oxidation of these susceptible residues and prevent irreversible loss of activity would be advantageous. The present study shows that the steady‐state redox potential of the cysteine/cystine couple (Eh = –145 mV) in cells is sufficiently oxidized (>90 mV) relative to the GSH/GSSG (–250 mV) and thioredoxin (Trx1, –280 mV) redox couples for the cysteine/cystine couple to function as an oxidant in redox switching. Consequently, the cysteine/cystine couple provides a means to oxidize proteins without direct involvement of more potent oxidants. A circuitry model incorporating cysteine as a redox node, along with Trx1 and GSH, reveals how selective interactions between the different thiol/disulfide couples and reactive protein thiols could differentially regulate metabolic functions. Moreover, inclusion of cysteine/cystine as a signaling node distinct from GSH and Trx1 significantly expands the redox range over which protein thiol/disulfide couples may operate to control physiologically relevant processes.

Human acetylator genotype: Relationship to colorectal cancer incidence and arylamine N-acetyltransferase expression in colon cytosol

Polymorphic expression of arylamine N-acetyltransferase (EC 2.3.1.5) may be a differential risk factor in metabolic activation of arylamine carcinogens and susceptibility to cancers related to arylamine exposures. Human epidemiological studies suggest that rapid acetylator phenotype may be associated with higher incidences of colorectal cancer. We used restriction fragment length polymorphism analysis to determine acetylator genotypes of 44 subjects with colorectal cancer and 28 non-cancer subjects of similar ethnic background (i.e., approximately 25% Black and 75% White). The polymorphic N-acetyltransferase gene (NAT2) was amplified by the polymerase chain reaction from DNA templates derived from human colons of colorectal and non-cancer subjects. No significant differences inNAT2 allelic frequencies (i.e., WT, M1, M2, M3 alleles) or in acetylator genotypes were found between the colorectal cancer and non-cancer groups. No significant differences inNAT2 allelic frequencies were observed between Whites and Blacks or between males and females. Cytosolic preparations from the human colons were tested for expression of arylamine N-acetyltransferase activity. Although N-acetyltransferase activity was expressed for each of the arylamines tested (i.e., p-aminobenzoic acid, 4-aminobiphenyl, 2-aminofluorene, β-naphthylamine), no correlation was observed between acetylator genotype and expression of human colon arylamine N-acetyltransferase activity. Similarly, no correlation was observed between subject age and expression of human colon arylamine N-acetyltransferase activity. These results suggest that arylamine N-acetyltransferase activity expressed in human colon is catalyzed predominantly by NAT1, an arylamine N-acetyltransferase that is not regulated byNAT2 acetylator genotype. The ability to determine acetylator genotype from DNA derived from human surgical samples should facilitate further epidemiological studies to assess the role of acetylator genotype in various cancers.

Maternal exposure to benzo[a]pyrene alters development of T lymphocytes in offspring.

Childhood cancer has been increasing significantly over the past two decades in the United States, suggesting that environmental exposures may be playing a causative role. One such cause may be maternal smoking during pregnancy. Suspected carcinogens in cigarette smoke and environmental pollution include N-nitrosamines and polycyclic aromatic hydrocarbons, which may be several micrograms per exposure. Previously, we have shown that mouse progeny of mothers exposed to benzo[a]pyrene (B[a]P) during midpregnancy had abnormalities in their humoral and cell-mediated immune response. Immunodeficiency was detectable during gestation, at one week after birth and persisted for 18 months. Tumor incidences in progeny were eight to 10-fold higher than in controls. The present study compared frequencies of CD4+, CD8+, V gamma 2+, and V beta 8+ T cells in progeny following in utero exposure to B[a]P. The significant reduction in newborn CD4+CD8+, CD4+CD8+V beta 8+ thymocytes and CD4+ splenocytes from 1-week-old progeny, suggests that B[a]P induces abnormal changes in developing T cells. These early alterations may lead to postnatal T cell suppression, thus providing a more suitable environment for the growth of tumors later in life. These results suggest that developmental immunosuppression mediated by B[a]P may play a critical role in the relationship between maternal exposures and childhood carcinogenesis.

Polymorphism of CYP2D6 in Black populations: implications for psychopharmacology

Drug-metabolizing enzymes found primarily in the liver (CYP450) are a major determinant of therapeutic drug response. Polymorphism dependent upon race/ethnic origin for CYP2D6 is now well-established. Despite consistent reports of ethnic differences in pharmacologic response to antidepressants and neuroleptics, there is a paucity of data on controlled clinical trials and studies determining polymorphic characteristics of CYP2D6 enzymes in African-Americans. There is little and conflicting information available on black populations (Africans, bushmen, Australian Aborigines or African Americans). The prevalence of poor metabolizers in Black populations has been estimated from 0 to 19%, compared with consistent reports of poor metabolizer status in Caucasians (5-10%) and Asians (0-2%). Within the extensive metabolizer category, Asians have higher metabolic ratios (that is, slower metabolism) than Caucasian extensive metabolizers. A high frequency of a mutant gene, CYP2D6*10 has been associated with the slower metabolic rate in Asians. Previous research suggests that slower metabolic rates compared with Caucasians may also be characteristic of Black populations. Recent reports suggest that a novel gene mutant in Black populations, CYP2D6*17, associated with a slower metabolic rate, may occur in a high frequency in these populations. Common clinical practice, supported by controlled clinical studies in Asians, have led to a reduction in dosage recommendations for many antidepressants and neuroleptics for this ethnic group. It is imperative that the determinants of bioavailability be established in African-Americans in order to establish rational drug therapy guidelines for this population.

Detection of DNA adducts in developing CD4+CD8+ thymocytes and splenocytes following in utero exposure to benzo[a]pyrene

ABSTRACT Environmental carcinogen exposure may play an important role in the incidence of cancer in children. In addition to environmental pollutants, maternal smoking during pregnancy may be a contributing factor. Major carcinogenic components of cigarette smoke and other combustion by-products in the environment include polycyclic aromatic hydrocarbons (PAH). Mouse offspring exposed during midpregnancy to the PAH, benzo[a]pyrene (B[a]P), show significant deficiencies in their immune functions, observed in late gestation which persist for at least 18 months. Tumor incidences in these progeny are 8 to 10-fold higher than in controls. We have demonstrated a significant reduction in thymocytes (CD4+CD8+, CD4+CD8+Vβ8+, CD4+CD8+Vγ2+) from newborn and splenocytes (CD4+CD8−) from 1-week-old mouse progeny exposed to B[a]P in utero. To investigate possible causes of the observed T cell reduction, we analyzed the thymocytes and splenocytes from progeny and maternal tissues for the presence of B[a]P-DNA adducts. Adducts were detected in maternal, placental and offspring lymphoid tissues at day 19 of gestation, at birth and 1-wk after birth. The presence of B[a]P-DNA adducts in immature T cells may, in part, explain the previously observed T cell immunosuppression and tumor susceptibility in mice exposed to B[a]P in utero. The effects of DNA lesions on progeny T cells may include interference with normal T-cell development. These results provide a possible explanation for the relationship between maternal smoking during pregnancy and childhood carcinogenesis.

Phytochemical induction of cell cycle arrest by glutathione oxidation and reversal by N-acetylcysteine in human colon carcinoma cells.

Cancer prevention by dietary phytochemicals has been shown to involve decreased cell proliferation and cell cycle arrest. However, there is limited understanding of the mechanisms involved. Previously, we have shown that a common effect of phytochemicals investigated is to oxidize the intracellular glutathione (GSH) pool. Therefore, the objective of this study was to evaluate whether changes in the glutathione redox potential in response to dietary phytochemicals was related to their induction of cell cycle arrest. Human colon carcinoma (HT29) cells were treated with benzyl isothiocyanate (BIT) (BIT), diallyl disulfide (DADS), dimethyl fumarate (DMF), lycopene (LYC) (LYC), sodium butyrate (NaB) or buthione sulfoxamine (BSO, a GSH synthesis inhibitor) at concentrations shown to cause oxidation of the GSH: glutathione disulfide pool. A decrease in cell proliferation, as measured by [ 3 H]-thymidine incorporation, was observed that could be reversed by pretreatment with the GSH precursor and antioxidant N-acetylcysteine (NAC). Cell cycle analysis on cells isolated 16 h after treatment indicated an increase in the percentage (ranging from 75–30% for benzyl isothiocyanate and lycopene, respectively) of cells at G2/M arrest compared to control treatments (dimethylsulfoxide) in response to phytochemical concentrations that oxidized the GSH pool. Pretreatment for 6 h with N-acetylcysteine (NAC) resulted in a partial reversal of the G2/M arrest. As expected, the GSH oxidation from these phytochemical treatments was reversible by NAC. That both cell proliferation and G2/M arrest were also reversed by NAC leads to the conclusion that these phytochemical effects are also mediated, in part, by intracellular oxidation. Thus, one potential mechanism for cancer prevention by dietary phytochemicals is inhibition of the growth of cancer cells through modulation of their intracellular redox environment.

Variation in the response of T cells to Concanavalin A after in vitro exposure to benzo[A]pyrene and 2-aminofluorene

The ability of the polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BP) and its metabolites to be immunosuppressive has been well documented by many investigators. The arylamine, 2-aminofluorene (AF) and its metabolic intermediates have not been as widely studied in this regard. Here, we investigate the effect of BP, 3-hydroxy-BP (3-OH-BP), AF, N-hydroxy-AF (N-OH-AF) and acetyl-AF (AAF) on T-cell proliferation using the T-cell mitogen, Concanavalin A (ConA). These compounds as well as BP-7, 8-diol-9, 10-epoxide (BPDE) were also used to determine their effect on T-cell-mitogen binding. Both AF and BP are substrates for the P-450 and flavin-containing monooxygenase enzyme system, which can be induced with beta-naphthoflavone (beta NF). We incubated beta nF with BP and AF to determine the effect of a P-450 inducer on BP and AF mediated-ConA suppression. Here we demonstrate that BP, 3-OH-BP, AF, and AAF are able to suppress the proliferative response to ConA, while N-OH-AF cannot. Further, we show that BP, 3-OH-BP, BPDE, AF and N-OH-AF do not alter the ability of ConA to bind the mitogen receptor of splenic T-cells, indicating an intracellular mechanism for suppression. Studies with beta NF indicate that this P-450 inducer enhances the anti-proliferative effect of BP, while it abolishes this effect of AF.

Overexpression of heat shock factor 1 inhibits butyrate-induced differentiation in colon cancer cells

Abstract Produced by dietary fiber, butyrate is a potential chemopreventive agent against colon cancer. It stimulates proliferation of normal colonic epithelial cells but induces growth inhibition, differentiation, apoptosis, or a combination of effects in colon carcinoma cells. In this study, we used cDNA membrane arrays and real-time reverse transcriptase– polymerase chain reaction to identify stress genes that were differentially regulated by sodium butyrate (NaB) in HT 29 human colon carcinoma cells. The results indicated that a group of heat shock protein (hsp) genes were upregulated by 3 mM NaB within the first 24 hours of exposure. Because the transcription of hsp genes is under the control of heat shock factors (HSFs), we measured the effects of overexpressed HSF-1 on the responses of HT 29 cells to NaB. Overexpression of HSF-1 inhibited NaB-induced differentiation as measured by alkaline phosphatase activity and carcinoembryonic antigen expression. These results suggest that increased expression of HSFs and Hsps might render colon carcinoma cells resistant to the chemopreventive effects of butyrate.

Abstract 1850: Benzyl isothiocyanate causes proteasome-mediated degradation of aryl hydrocarbon receptor in Hepa1c1c7 cells

The biotransformation and detoxification of polycyclic aromatic carcinogens is dependent upon the activity of CYP1A1 and NQO1. Transcriptional activation of these enzymes is regulated by the aryl hydrocarbon receptor (AhR) and Nuclear Factor-Erythroid 2-Related Factor. These transcription factors reside in the cytoplasm and when activated, shed their respective chaperones and translocate into the nucleus where they interact with nuclear proteins involved with transcription of genes possessing xenobiotic (XRE) and antioxidant (ARE) response elements. We have previously reported that the phytochemical benzyl isothiocyanate (BITC) induces ligand-independent nuclear translocation of the AhR in Hepa1c1c7 cells and also induces AhR heterodimerization with ARNT. This suggests that BITC might bind the AhR and affect enzyme activities through transcriptional regulation of XRE-responsive genes. We also have observed that BITC causes rapid degradation of the AhR both alone and in combination with known receptor ligands. In the current study, we investigated whether BITC causes proteasomal degradation of the AhR and whether this degradation occurs through increased ubiqitination of the receptor. This is important because it provides a potential mechanism by which BITC affects AhR turnover and consequently its regulation of phase I and II enzymes. Hepa1 cells were treated overnight with DMSO (control), 10 µM BITC, 10 µM β-napthoflavone (BNF) or a combination of BITC and BNF both at 10 µM. AhR was immunoprecipitated and detected using western blot. AhR was degraded in samples treated with BITC alone compared to control. Additionally, BITC enhanced degradation of AhR in samples treated with both BITC and BNF compared to BNF alone. Western blots re-probed with anti-ubiquitin showed increased ubiquitination of proteins in BITC and BNF-treated samples compared to control. In additional experiments, cells were treated for 16 hours with DMSO, 10 µM BITC, 10 µM BNF or a combination of BNF (10 µM) and increasing concentrations of BITC (1-10 µM). Sub-cellular fractionation of samples treated with a combination of BITC and BNF showed a dose-dependent increase in the amount of insoluble AhR compared to either BITC or BNF alone. AhR degradation was blocked in cells pre-treated with the proteasome inhibitor MG132 but not in cells pre-treated with broad spectrum protease inhibitors or PMSF. This suggests that degradation is specific to the proteasome rather than other proteases such as serine proteases and calpains. Collectively these results show that BITC degrades the AhR and this is mediated by ubiquitin and the proteasome. AhR degradation might affect transcription of XRE responsive genes and therefore carcinogen activation. These results also provide a potential mechanism for BITC as a cancer chemopreventive agent. (Support: NIH MBRS/GM028248; RCMI RR033034) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1850. doi:10.1158/1538-7445.AM2011-1850