Transgenic expression of aflatoxin aldehyde reductase (AKR7A1) modulates aflatoxin B1 metabolism but not hepatic carcinogenesis in the rat

Abstract

In both experimental animals and humans, aflatoxin B(1) (AFB(1)) is a potent hepatic toxin and carcinogen against which a variety of antioxidants and experimental or therapeutic drugs (e.g., oltipraz, related dithiolethiones, and various triterpenoids) protect from both acute toxicity and carcinogenesis. These agents induce several hepatic glutathione S-transferases (GST) as well as aldo-keto reductases (AKR) which are thought to contribute to protection. Studies were undertaken in transgenic rats to examine the role of one inducible enzyme, AKR7A1, for protection against acute and chronic actions of AFB(1) by enhancing detoxication of a reactive metabolite, AFB(1) dialdehyde, by reduction to alcohols. The AFB(1) dialdehyde forms adducts with protein amino groups by a Schiff base mechanism and these adducts have been theorized to be at least one cause of the acute toxicity of AFB(1) and to enhance carcinogenesis. A liver-specific AKR7A1 transgenic rat was constructed in the Sprague-Dawley strain and two lines, AKR7A1(Tg2) and AKR7A1(Tg5), were found to overexpress AKR7A1 by 18- and 8-fold, respectively. Rates of formation of AFB(1) alcohols, both in hepatic cytosols and as urinary excretion products, increased in the transgenic lines with AKR7A1(Tg2) being the highest. Neither line offered protection against acute AFB(1)-induced bile duct proliferation, a functional assessment of acute hepatotoxicity by AFB(1), nor did they protect against the formation of GST-P positive putative preneoplastic foci as a result of chronic exposure to AFB(1). These results imply that the prevention of protein adducts mediated by AKR are not critical to protection against AFB(1) tumorigenicity.

FIG. 1.

Schematic of enzymatic and chemical conversion of AFB₁ to the reactive dialdehyde and its reduction by AKR7A1 to three possible aflatoxin alcohols.

FIG. 2.

Transgene expression of AKR7A1. (A) The DNA construct was designed for liver-specific expression using the pLiv-7 vector containing sequences from the human APOE gene: 3 kb of 5′-flanking region (black); the first exon (E1, dotted), first intron (open), and first six nucleotides of the second exon (E2, dotted); 0.25 kb of 3′-flanking region including the polyadenylation signal (dashed) and a 1.7-kb hepatic control region of the APOE/C-I gene locus (gray). The AKR7A1 cDNA was inserted into the polylinker region using the KpnI and XhoI restriction sites. (B) Transgene expression of AKR7A1. Protein levels of AKR7A1 in samples of liver cytosol (30 μg protein/lane) from one male (M) and one female (F) of four separate AKR7A1 transgenic rat lines were analyzed by immunoblot. The samples, identified at the top, are nontransgenic genetic control animals (neg), transgenic animals (pos), and nontransgenic rats pretreated with 3H-1,2-dithiole-3-thione (D3T) a known inducer of AKR7A1.

FIG. 3.

(A) Formation of the C-6a monoalcohol, C-8 monoalcohol and dialcohol following incubation of hepatic cytosols with aflatoxin dialdehyde. Incubations were conducted for 3 min with AFB₁ dialdehyde susbtrate (7μM). All three AFB₁ alcohols could be detected in nontransgene hepatic cytosols under nonlinear incubation conditions (10μM substrate, 10 min). Metabolites were quantified by isotope dilution mass spectrometry. *p < 0.05, AKR7A1^Tg2 compared to AKR7A1^Tg5 by ANOVA. (B) GST activities in hepatic cytosols using chlorodinitrobenzene as substrate. *p < 0.05, D3T compared to vehicle treated by ANOVA. Values are mean ± SE (N = 3).

FIG. 4.

Urinary excretion of AFB₁ dialcohol levels in AKR7A1 transgenic and D3T-treated nontransgenic rats. AFB₁ dialcohol was quantified by isotope dilution mass spectrometry as described in “Materials and Methods” and in reference 24. Values are mean ± SE (N = 3).

FIG. 5.

Evaluation of the AKR7A1 transgene on AFB₁-induced GST-P positive foci formation. At 5 weeks of age, litter mates expressing AKR7A1 and genetic controls were orally gavaged with 25 μg AFB₁ 5 days per week for 4 successive weeks. The livers were removed 6 weeks later, fixed in acetone, and immunohistochemically stained using an antibody recognizing GST-P. The number and size of the GST-P positive foci were measured by light microscopy and the volume % of GST-P positive foci (analogous to tumor burden) calculated. There were no statistically significant differences between AKR7A1 positive and genetic control. One of 12 rats with an unusually high focal burden (74%) accounted for the high group mean and large SE in the male AKR7A1^Tg2 group. These scatter plots are accompanied with median bars (N ranged from 12 to 16 rats per group for the AKR7A1^Tg2 and six to eight rats per group for the AKR7A1^Tg5 rats).

FIG. 6.

Influence of AKR transgene on AFB₁ hepatic DNA and serum albumin protein adduct levels. AFAR^Tg2 positive animals had statistically significant lower levels of AFB₁-lysine adducts compared to their genetic control littermates (p < 0.05). D3T-treated animals had statistically significant lower levels of AFB₁ N⁷-guanine adducts compared to vehicle treated (*p < 0.05). Quantification for data shown was by isotope dilution mass spectrometry. Statistical comparisons were achieved by ANOVA. Values are mean ± SE (N = 3).