Sulforaphane- and phenethyl isothiocyanate-induced inhibition of aflatoxin B1-mediated genotoxicity in human hepatocytes: role of GSTM1 genotype and CYP3A4 gene expression - PubMed (original) (raw)

Sulforaphane- and phenethyl isothiocyanate-induced inhibition of aflatoxin B1-mediated genotoxicity in human hepatocytes: role of GSTM1 genotype and CYP3A4 gene expression

Kerstin Gross-Steinmeyer et al. Toxicol Sci. 2010 Aug.

Abstract

Primary cultures of human hepatocytes were used to investigate whether the dietary isothiocyanates, sulforaphane (SFN), and phenethyl isothiocyanate (PEITC) can reduce DNA adduct formation of the hepatocarcinogen aflatoxin B(1) (AFB). Following 48 h of pretreatment, 10 and 50 microM SFN greatly decreased AFB-DNA adduct levels, whereas 25muM PEITC decreased AFB-DNA adducts in some but not all hepatocyte preparations. Microarray and quantitative reverse transcriptase (RT)-PCR analyses of gene expression in SFN and PEITC-treated hepatocytes demonstrated that SFN greatly decreased cytochrome P450 (CYP) 3A4 mRNA but did not induce the expression of either glutathione S-transferase (GST) M1 or GSTT1. The protective effects of SFN required pretreatment; cotreatment of hepatocytes with SFN and AFB in the absence of pretreatment had no effect on AFB-DNA adduct formation. When AFB-DNA adduct formation was evaluated by GST genotype, the presence of one or two functional alleles of GSTM1 was associated with a 75% reduction in AFB-DNA adducts, compared with GSTM1 null. In conclusion, these results demonstrate that the inhibition of AFB-DNA adduct formation by SFN is dependent on changes in gene expression rather than direct inhibition of catalytic activity. Transcriptional repression of genes involved in AFB bioactivation (CYP3A4 and CYP1A2), but not transcriptional activation of GSTs, may be responsible for the protective effects of SFN. Although GSTM1 expression was not induced by SFN, the presence of a functional GSTM1 allele can afford substantial protection against AFB-DNA damage in human liver. The downregulation of CYP3A4 by SFN may have important implications for drug interactions.

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Figures

FIG. 1.

FIG. 1.

Effects of SFN and PEITC on AFB-DNA adduct formation in human hepatocytes. Hepatocytes were treated with two concentrations of each phytochemical for 48 h and subsequently coincubated with 0.4μM 3H-AFB and phytochemical. Control represents nonmodulated AFB-DNA adduct level, where cells were treated with vehicle instead of phytochemical. AFB-DNA adduct levels are expressed as percentages of control and represent means and SEs from six independent experiments (i.e., hepatocytes from six donors). The 100% control value for six hepatocyte preparations was 5.9 adducts per 107 nucleotides (range 2.2–10.7 adducts per 107 nucleotides). “*” Denotes 0.01 < p < 0.05; “**” denotes p < 0.01 comparing vehicle control (100%) versus phytochemical incubations.

FIG. 2.

FIG. 2.

Effects of SFN on CYP3A4 mRNA expression in cultured human primary hepatocytes over 96 h. CYP3A4 mRNA levels of hepatocytes were determined at the following times and treatments: (A) at 0 h (no DMSO or SFN treatment); (B) following 24 h treatment with 10 or 25μM SFN or DMSO; (C) following 48 h of SFN or DMSO treatment; (D) 48 h in culture with DMSO (no SFN) followed by 24 h of SFN or DMSO treatment; (E) 48 h in culture with DMSO (no SFN) followed by 48 h of SFN or DMSO treatment. Bars represent means of normalized CYP3A4 mRNA expression, including corresponding SD from three individual cell culture vessels from a single hepatocyte donor. “*” Denotes p < 0.05 comparing vehicle control versus SFN incubations within each condition.

FIG. 3.

FIG. 3.

Modulation of AFB-DNA adduct formation by SFN in cultured human primary hepatocytes at different treatment conditions. To assess contribution of transcriptional versus enzyme inhibition effects, we measured AFB-DNA adduct levels under three different conditions: (1) following a 48-h pretreatment with SFN, cells were incubated for 6 h in media containing 3H-AFB but no SFN (to detect transcriptional effects); (2) following a 48-h pretreatment with SFN, cells were incubated for an additional 6 h in media containing 3H-AFB and SFN (to detect both transcriptional effects and enzyme inhibition effects); (3) following a 48-h treatment with vehicle only (no SFN), cells were incubated for an additional 6 h in media containing 3H-AFB and SFN (to detect enzyme inhibition effects). SFN was applied at 10μM. Conditions 1, 2, and 3 were compared with an analogous treatment without SFN application, which served as control (equal to 100%), for example, following a 48-h treatment with vehicle only (no SFN), cells were incubated for an additional 6 h in media containing 3H-AFB only (no SFN). AFB-DNA adduct levels are expressed as percentages of control and represent means and SDs from three independent experiments (e.g., hepatocytes from three individual preparations). Conditions 1 and 2 were not significantly different from each other, but each was different from control (p < 0.05), marked as “*.” Condition 3 was significantly different from conditions 1 and 2 (p < 0.05) but not from the control (#).

FIG. 4.

FIG. 4.

Modulation of AFB-DNA adduct formation in the context of the GSTM1 genotype status. A total of 11 different hepatocyte preparations were examined for AFB-DNA binding in the presence or absence of 10μM SFN pretreatment. Six of the samples were GSTM1 null and five were GSTM1 positive. AFB-DNA adducts per 107 nucleotides were calculated and are shown. Each bar represents the mean and SEM. ANOVA indicated variances that were not statistically different among the groups. Statistical significance was determined by unpaired _t_-test with equal variances. All groups were significantly different (p < 0.05), with p values for individual comparisons shown.

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