Increased resistance of tumor cells to daunorubicin after transfection of cDNAs coding for anthracycline inactivating enzymes

Carbonyl reduction is a main but undesired metabolic pathway of the anti-cancer drug daunorubicin (DRC). The resulting alcohol metabolite daunorubicinol has a far less anti-tumor potency and, in addition, is responsible for the life-threatening cardiac toxicity that limits the clinical use of DRC. Elevated levels of carbonyl-reducing enzymes in cancer cells may therefore contribute to the development of DRC chemoresistance and affect the clinical outcome. In the present investigation, human pancreas carcinoma cells were transfected with three important DRC reductases, namely carbonyl reductase (CBR1), aldehyde reductase (AKR1A1) and aldose reductase (AKR1B1), and levels of resistance towards DCR determined. Overexpression of all three reductases lead to a higher DRC inactivation and to an elevation of chemoresistance (7-fold for CBR1, 4.5-fold for AKR1A1 and 3.7-fold for AKR1B1), when IC(50)-values were considered. Coadministration of DRC reductase inhibitors in DRC chemotherapy may be desirable since this would reduce the formation of the cardiotoxic alcohol metabolite and prevent drug resistance.     

Mechanism of action of dietary chemoprotective agents in rat liver: induction of phase I and II drug metabolizing enzymes and aflatoxin B1 metabolism

A range of potential chemoprotective agents, most of them natural dietary constituents, has been examined for ability to modulate both phase I (cytochrome P450 1A1, 1A2, 2B1/2, 2C11, 2E1, 3A, 4A) and phase II drug metabolizing enzymes (glutathione S-transferases, in particular subunits Yc2 and P, aflatoxin B1-aldehyde reductase and quinone reductase) in rat liver. In addition to assays of total enzyme activity and Western blots for individual isozymes, the ability of microsomes to metabolize aflatoxin B1, and of cytosols to conjugate aflatoxin B1 (AFB1)-epoxide to GSH and to produce AFB1-dialcohol, were measured. Induction of gamma-glutamyl transpeptidase activity was examined by histochemistry. Differing patterns of induction were observed, reflecting differences in the control of expression of the individual enzymes studied. Of the compounds examined, butylated hydroxytoluene, ethoxyquin, indole-3-carbinol and phenethyl isothiocyanate were the most potent bifunctional agents (inducing both phase I and II activities). Oltipraz, while only weakly inducing CYP1A2 and 2B1/2, was a potent inducer of phase II enzymes. Caffeic acid, garlic oil, sinigrin and propyl gallate all showed some ability to induce phase II enzymes. 4-Methyl catechol, alpha-tocopherol and red wine decreased certain phase I enzyme activities, while inducing total GST activity. Butylated hydroxytoluene, ethoxyquin, garlic oil and indole-3-carbinol induced gamma glutamyltranspeptidase in periportal hepatocytes. Particularly because of their ability to induce the detoxifying activities of glutathione S-transferase Yc2 and aldehyde reductase, butylated hydroxytoluene, ethoxyquin, indole-3-carbinol, oltipraz, phenethyl isothiocyanate and sinigrin will be effective blocking agents in rodents, if administered prior to AFB1. While these studies indicate the relative contributions of phase I and II metabolism in the overall protective effect in rat, care should be taken that a similar balance is achieved in man, and that relevant enzymes or iso forms are induced.      

Mechanisms of protection against aflatoxin B(1) genotoxicity in rats treated by organosulfur compounds from garlic

Diallyl sulfide (DAS) and diallyl disulfide (DADS), two garlic constituents, were found previously to inhibit aflatoxin B(1) (AFB(1))-initiated carcinogenesis in rat liver, DADS being the most effective. In order to study the mechanisms involved in this protection, we have examined the ability of liver microsomes and cytosols from DAS- and DADS-treated rats to modulate the mutagenicity and the metabolism of AFB(1). We also examined the effects of these compounds on the expression of cytochromes P450 (CYP) and phase II enzymes known to be involved in AFB(1) metabolism. Administration of DAS (1 mmol/kg for 4 days) to rats resulted in significant inhibition of microsome-mediated mutagenicity of AFB(1), whereas DADS treatment did not alter AFB(1) mutagenicity. DAS treatment increased the metabolism of AFB(1) mainly towards the formation of AFQ(1) and AFM(1), which might account for the reduction of AFB(1) microsomal-mediated mutagenicity. DADS treatment slightly affected the oxidative metabolism of AFB(1). DAS and DADS induced CYP3A2, CYP2B1 and CYP2B2, DAS being more potent. Cytosols from DAS- and DADS-treated rats produced a significant inhibition of AFB(1)-8,9-epoxide (AFBO)-induced mutagenicity and significantly increased the cytosolic formation of AFB(1)-glutathione conjugates, DADS treatment being more effective. Western blot analysis showed that DADS is a potent inducer of glutathione S-transferase A5 (rGSTA5) and AFB(1) aldehyde reductase 1 (rAFAR1), while DAS is a weak inducer of these enzymes. Finally, we demonstrated that antibodies raised against rGSTA5 strongly reduced the antimutagenic activity of cytosols from DAS- and DADS-treated rats against AFBO. All together, these results demonstrate that DAS prevents AFB(1) mutagenicity through a dual mechanism, i.e. by modulating both the phase I and II metabolism of AFB(1), whereas DADS acts mainly by increasing the phase II metabolism of AFB(1). The induction of rGSTA5 and rAFAR1 is probably the main mechanism by which allyl sulfides give protection against AFB(1)-induced carcinogenesis.     

Glutathione S-transferase mu in human lymphocyte and liver: role in modulating formation of carcinogen-derived DNA adducts.

Glutathione transferase (GT) activity towards trans-stilbene oxide (tSBO), benzo[a]pyrene-4,5-oxide (B[a]PO) and 1-chloro-2,4-dinitrobenzene (CDNB) was measured in human liver and lymphocytes. GT-tSBO activity is catalyzed by GT mu which has polymorphic expression in human lymphocytes. Our results show that activity of GT-tSBO in lymphocytes correlates with its activity in liver (r = 0.7, P less than 0.001). GT activity towards BPO (GT-BPO) also correlated with GT-tSBO in lymphocytes and liver. However, interindividual variation of GT-BPO is less than that of GT-tSBO, suggesting that BPO may not be as specific a substrate for GT mu and therefore other GT isozymes may contribute to BPO conjugation. Conjugation of CDNB by GT was not different using cytosols from either high or low GT mu individuals. The functional significance of the GT-mu polymorphism was evaluated by measuring its effect on benzo[a]pyrene (B[a]P)- and aflatoxin B1 (AFB1)-DNA adduct formation in vitro. Human liver cytosols prepared from persons having low or high GT-tSBO activity were incubated with human liver microsomes, calf thymus DNA and B[a]P or AFB1. HPLC analysis revealed that the major B[a]P adduct was dG(N2)-7 beta, 8 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-dG). BPDE-dG adducts were decreased equally by cytosols from either low or high conjugators. In contrast, AFB1-DNA binding was inhibited to a greater extent in high conjugators than low conjugators. HPLC analysis demonstrates that adducts formed were AFB1-FAPyr and AFB1-N7-Gua. The correlation between AFB1-DNA adduct concentrations and GT mu activity was highly significant with a correlation coefficient of r = 0.88 at P less than 0.001. These results suggest that GT mu plays an important role in detoxifying DNA reactive metabolites of AFB1 and this enzyme may be a susceptibility marker for AFB1 related liver cancer. Moreover, our data demonstrate that lymphocytes are a reliable surrogate tissue for detecting liver GT mu polymorphisms.     

Chemoprevention of aflatoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer of aflatoxin B1-aldehyde reductase, the glutathione S-transferase A5 and P1 subunits, and NAD(P)H:quinone oxidoreductase in rat liver

Structurally diverse compounds can confer resistance to aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Treatment with either phytochemicals [benzyl isothiocyanate, coumarin (CMRN), or indole-3-carbinol] or synthetic antioxidants and other drugs (butylated hydroxyanisole, diethyl maleate, ethoxyquin, beta-naphthoflavone, oltipraz, phenobarbital, or trans-stilbene oxide) has been found to increase hepatic aldo-keto reductase activity toward AFB1-dialdehyde and glutathione S-transferase (GST) activity toward AFB1-8,9-epoxide in both male and female rats. Under the conditions used, the natural benzopyrone CMRN was a major inducer of the AFB1 aldehyde reductase (AFAR) and the aflatoxin-conjugating class-alpha GST A5 subunit in rat liver, causing elevations of between 25- and 35-fold in hepatic levels of these proteins. Induction was not limited to AFAR and GSTA5: treatment with CMRN caused similar increases in the amount of the class-pi GST P1 subunit and NAD(P)H: quinone oxidoreductase in rat liver. Immunohistochemistry demonstrated that the overexpression of AFAR, GSTA5, GSTP1, and NAD(P)H:quinone oxidoreductase affected by CMRN is restricted to the centrilobular (periacinar) zone of the lobule, sometimes extending almost as far as the portal tract. This pattern of induction was also observed with ethoxyquin, oltipraz, and trans-stilbene oxide. By contrast, induction of these proteins by beta-naphthoflavone and diethyl maleate was predominantly periportal. Northern blotting showed that induction of these phase II drug-metabolizing enzymes by CMRN was accompanied by similar increases in the levels of their mRNAs. To assess the biological significance of enzyme induction by dietary CMRN, two intervention studies were performed in which the ability of the benzopyrone to inhibit either AFB1-initiated preneoplastic nodules (at 13 weeks) or AFB1-initiated liver tumors (at 50 weeks) was investigated. Animals pretreated with CMRN for 2 weeks prior to administration of AFB1, and with continued treatment during exposure to the carcinogen for a further 11 weeks, were protected completely from development of hepatic preneoplastic lesions by 13 weeks. In the longer-term dietary intervention, treatment with CMRN before and during exposure to AFB1 for a total of 24 weeks was found to significantly inhibit the number and size of tumors that subsequently developed by 50 weeks. These data suggest that consumption of a CMRN-containing diet provides substantial protection against the initiation of AFB1 hepatocarcinogenesis in the rat.     

Genetic variation of Aflatoxin B1 aldehyde reductase genes (AFAR) in human tumour cells

AFAR genes play a key role in the detoxification of the carcinogen Aflatoxin B(1) (AFB(1)). In the rat, Afar1 induction can prevent AFB(1)-induced liver cancer. It has been proposed that AFAR enzymes can metabolise endogenous diketones and dialdehydes that may be cytotoxic and/or genotoxic. Furthermore, human AFAR1 catalyses the rate limiting step in the synthesis of the neuromodulator gamma-hydroxybutyrate (GHB) and was found elevated in neurodegenerative diseases such as Alzheimer's and dementia with Lewy bodies (DLB). The human AFAR gene family maps to a genomic region in 1p36 of frequent hemizygous deletions in various human cancers. To investigate, if genetic variation of AFAR1 and AFAR2 exists that may alter protein detoxification capabilities and confer susceptibility to cancer, we have analysed a spectrum of human tumours and tumour cell lines for genetic heterogeneity. From 110 DNA samples, we identified nine different amino acid changes; two were in AFAR1 and seven in AFAR2. In AFAR1, we found genetic variation in the proposed substrate-binding amino acid 113, encoding Ala(113) or Thr(113). An AFAR2 variant had a Glu(55) substituted by Lys(55) at a position that is conserved among many aldo-keto reductases. This polarity change may have an effect on the proposed substrate binding amino acids nearby (Met(47), Tyr(48), Asp(50)). Further population analyses and functional studies of the nine variants detected may show if these variants are disease-related.     

AKR7A3 modulates the metastasis of pancreatic ductal adenocarcinoma through regulating PHGDH-suppressed autophagy

AKR7A3 is a member of the aldo-keto reductase (AKR) protein family, whose primary purpose is to reduce aldehydes and ketones to generate primary and secondary alcohols. It has been reported that AKR7A3 is downregulated in pancreatic cancer (PC). However, the mechanism underlying the effects of AKR7A3 in PC remains largely unclarified. Here, we explored the biological function, molecular mechanism and clinical relevance of AKR7A3 in pancreatic ductal adenocarcinoma (PDAC). AKR7A3 expression was downregulated in PDAC compared with adjacent normal tissues, and the lower AKR7A3 expression was related to poor prognosis. In addition, our results demonstrated that AKR7A3 could be a potential diagnostic marker for PDAC, especially in the early stages. Knockdown of AKR7A3 promoted PDAC progression and chemoresistance, while inhibiting autophagy flux. Mechanistically, AKR7A3 affected the metastasis, autophagy, and chemoresistance of PDAC by regulating PHGDH. Overall, the present study suggests that AKR7A3 inhibits PDAC progression by regulating PHGDH-induced autophagy. In addition, AKR7A3 inhibits chemoresistance via regulating PHGDH and may serve as a new therapeutic target for PDAC.     

Reversal of inflammation-associated dihydrodiol dehydrogenases (AKR1C1 and AKR1C2) overexpression and drug resistance in nonsmall cell lung cancer cells by wogonin and chrysin

Dihydrodiol dehydrogenase (DDH) is a member of the aldo-keto reductases superfamily (AKR1C1-AKR1C4), which plays central roles in the metabolism of steroid hormone, prostaglandin and xenobiotics. We have previously detected overexpression of DDH as an indicator of poor prognosis and chemoresistance in human non-small lung cancer (NSCLC). We also found DDH expression to be closely related to chronic inflammatory conditions. The aim of this study was to investigate the links between inflammation, DDH expression and drug resistance in NSCLC cells. We showed that pro-inflammatory mediators including interleukin-6 (IL-6) could induce AKR1C1/1C2 expression in NSCLC cells and increase cellular resistance to cisplatin and adriamycin. This effect was nullified by Safingol, a protein kinase C inhibitor. Moreover, the expression of AKR1C1/1C2 was inversely correlated to NBS1 and apoptosis-inducing factor (AIF). We also showed that IL-6-induced AKR1C1/1C2 expression and drug resistance were inhibited by wogonin and chrysin, which are major flavonoids in Scutellaria baicalensis, a widely used traditional Chinese and Japanese medicine. In conclusion, this study demonstrated novel links of pro-inflammatory signals, AKR1C1/1C2 expression and drug resistance in NSCLC. The protein kinase C pathway may play an important role in this process. Overexpression of AKR1C1/1C2 may serve as a marker of chemoresistance. Further studies are warranted to evaluate wogonin and chrysin as a potential adjuvant therapy for drug-resistant NSCLC, especially for those with AKR1C1/1C2 overexpression.     

Human cytosolic enzymes involved in the metabolic activation of carcinogenic aristolochic acid: evidence for reductive activation by human NAD(P)H:quinone oxidoreductase

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Understanding which human enzymes are involved in AA metabolism is important in the assessment of an individual's susceptibility to this carcinogen. Using the 32P-postlabeling assay we examined the ability of enzymes of cytosolic samples from 10 different human livers and from one human kidney to activate the major component of the plant extract AA, 8-methoxy- 6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAI), to metabolites forming adducts in DNA. Cytosolic fractions of both organs generated AAI-DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(Deoxyadenosin-N6-yl)aristolactam I, 7-(deoxyguanosin-N2-yl)aristolactam I and 7-(deoxyadenosin-N6-yl)aristolactam II, indicating a possible demethoxylation reaction of AAI, were identified as AA-DNA adducts formed from AAI by all human hepatic and renal cytosols. To define the role of human cytosolic reductases in the activation of AAI, we investigated the modulation of AAI-DNA adduct formation by cofactors or selective inhibitors of the NAD(P)H:quinone oxidoreductase (NQO1), xanthine oxidase (XO) and aldehyde oxidase. We also determined whether the activities of NQO1 and XO in different human hepatic cytosolic samples correlated with the levels of AAI-DNA adducts formed by the same cytosolic samples. Based on these studies, we attribute most of the activation of AA in human cytosols to NQO1, although a role of cytosolic XO cannot be ruled out. With purified NQO1 from rat liver and kidney and XO from buttermilk, the major role of NQO1 in the formation of AAI-DNA adducts was confirmed. The orientation of AAI in the active site of human NQO1 was predicted from molecular modeling based on published X-ray structures. The results demonstrate for the first time the potential of human NQO1 to activate AAI by nitroreduction.     

Effect of microsomally activated AFB1 on GGT activity in 3 rat liver cell lines

Three cell lines derived from adult rat liver have been used to study changes in levels of gamma-glutamyl transferase (GGT), a possible marker for premalignant transformation in liver in vivo. None of the cell lines was able to metabolize aflatoxin B1 (AFB1) and treatment with AFB1 alone did not influence GGT activity. However, treatment with microsomally activated AFB1 increased the level of activity in a cell line (BL8L) derived from normal liver with very low levels of GGT, by as much as 10-fold, and 5-fold in a cell line (ARL) also isolated from normal rat liver, but which had subsequently undergone spontaneous transformation. Microsomes from rats pretreated with phenobarbitone were compared with those from 3-methylcholanthrene-treated animals. AFB1 activated by the former produced larger increases in GGT activity, but in no case did the enzyme levels approach that in a cell line (JBI) derived from a hepatoma in the liver of an AFB1-fed rat. Treatment of JBI cells with microsomally activated AFB1 produced no further increase in activity. Histochemical staining indicated an uneven distribution of enzyme in all cell populations, both before and after treatment. This cell-culture system is useful for further studies on the role of GGT in carcinogenesis.     

Bioactivation of aflatoxin B1 by lipoxygenases, prostaglandin H synthase and cytochrome P450 monooxygenase in guinea-pig tissues.

In the present investigation, we have examined the role of lipoxygenases in the bioactivation of aflatoxin B1 (AFB1) in hepatic and extrahepatic tissues. The enzyme activities were evaluated by determining [3H]AFB1-DNA adduct formation. The results demonstrated that both purified soybean lipoxygenase and guinea-pig tissue cytosolic lipoxygenases were able to activate AFB1 to form [3H]AFB1-DNA adduct(s). The reaction was completely inhibited by nordihydroguaiaretic acid (NDGA, 0.1 mM), a lipoxygenase inhibitor and an antioxidant, but not by indomethacin (0.1 mM), an inhibitor of prostaglandin H synthase (PHS), indicating that this reaction is associated with lipoxygenase activity, and/or is involved in a peroxyl radical process. While purified lipoxygenase showed arachidonic acid (AA)-dependent properties, the omission of AA did not diminish guinea-pig tissue cytosolic [3H]AFB1-DNA adduct formation, possibly because AA was released from lipid particles by AFB1. Within the range of hemoglobin (Hb) concentrations found in lung, kidney and liver cytosols (1.4-11.1 microM) and microsomes (0-0.5 microM), neither pure Hb, nor Hb of cytosols or microsomes from whole blood caused detectable AA-dependent AFB1-DNA binding. This indicates that Hb, as a contaminant with quasi-lipoxygenase activity, did not contribute to AFB1 activation attributed to guinea-pig tissue lipoxygenases. [3H]AFB1 concentrations at half-maximal DNA binding rate of pulmonary cytochrome P450 monooxygenases (P450) and lipoxygenases were similar, though P450 had a much higher maximum DNA binding rate. Pulmonary microsomal PHS activity for AFB1 activation was too low for its half-maximal binding concentrations of [3H]AFB1 and maximum rate to be accurately determined. In kidney, maximum rates for lipoxygenase, PHS and P450 were similar, whereas half-maximal binding concentrations for reactions by lipoxygenase and P450 were lower compared to that of PHS. The half-maximal binding concentration of hepatic lipoxygenase was significantly lower than those for PHS and P450. Hepatic half-maximal binding concentrations for PHS and P450 were similar, though P450 had a much higher maximum rate than PHS and lipoxygenases. These data suggest that lipoxygenase-catalyzed AFB1 activation can occur at low AFB1 concentrations. This may be important in view of human exposure to low AFB1 concentrations and predominant lipoxygenase activity in human airway epithelial cells. When expressed per gram of tissue, renal and hepatic PHS activities and renal lipoxygenase activities for AFB1 activation were similar, and higher than the activity of pulmonary PHS, while pulmonary PHS activity for the oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was similar to that in liver and lower than that in kidney.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pre-clinical activity of PR-104 as monotherapy and in combination with sorafenib in hepatocellular carcinoma

PR-104 is a clinical stage bioreductive prodrug that is converted in vivo to its cognate alcohol, PR-104A. This dinitrobenzamide mustard is reduced to activated DNA cross-linking metabolites (hydroxylamine PR-104H and amine PR-104M) under hypoxia by one-electron reductases and independently of hypoxia by the 2-electron reductase aldo-keto reductase 1C3 (AKR1C3). High expression of AKR1C3, along with extensive hypoxia, suggested the potential of PR-104 for treatment of hepatocellular carcinoma (HCC). However, a phase IB trial with sorafenib demonstrated significant toxicity that was ascribed in part to reduced PR-104A clearance, likely reflecting compromised glucuronidation in patients with advanced HCC. Here, we evaluate the activity of PR-104 in HCC xenografts (HepG2, PLC/PRF/5, SNU-398, Hep3B) in mice, which do not significantly glucuronidate PR-104A. Cell line differences in sensitivity to PR-104A in vitro under aerobic conditions could be accounted for by differences in both expression of AKR1C3 (high in HepG2 and PLC/PRF/5) and sensitivity to the major active metabolite PR-104H, to which PLC/PRF/5 was relatively resistant, while hypoxic selectivity of PR-104A cytotoxicity and reductive metabolism was greatest in the low-AKR1C3 SNU-398 and Hep3B lines. Expression of AKR1C3 in HepG2 and PLC/PRF/5 xenografts was in the range seen in 21 human HCC specimens. PR-104 monotherapy elicited significant reductions in growth of Hep3B and HepG2 xenografts, and the combination with sorafenib was significantly active in all 4 xenograft models. The results suggest that better-tolerated analogs of PR-104, without a glucuronidation liability, may have the potential to exploit AKR1C3 and/or hypoxia in HCC in humans.     

Comparative ultrastructural effects of aflatoxin B1 on mouse, rat, and human hepatocytes in primary culture

Human, rat, and mouse hepatocytes in primary culture were treated with aflatoxin B1 (AFB1) and examined for ultrastructural alterations. As early as 1 h following in vitro exposure to AFB1, there were ultrastructural changes in the nuclei of rat and human hepatocytes. The most prominent change in the nuclei was a segregation of nucleolar components that resembled the segregation in liver cells of rats exposed to AFB1 in vivo. The nucleolar segregations were developed by incubating rat hepatocytes for 24 h in a medium containing as little as 0.01 micrograms of AFB1 per ml. The minimum concentration to induce the same change in human hepatocytes was 0.1 micrograms/ml. No distinct nucleolar alteration was observed in mouse hepatocytes incubated in a medium containing 10 micrograms of AFB1 per ml. Irregular nuclear chromatin condensation also developed in the cells exposed to a higher concentration of AFB1, whereas little damage was observed in mitochondria and lysosomes. The similarity in morphological changes between our in vitro model and in vivo models previously investigated indicates that the hepatocytes in primary culture maintain the biological properties necessary for carcinogen responses similar to liver cells in vivo. In addition, the morphological changes in cultured rat and mouse hepatocytes induced by AFB1 correlate with in vivo experiments insofar as mice are relatively resistant, whereas rats are sensitive to AFB1 carcinogenesis. Thus, cultured hepatocyte systems may be a valuable tool to study genetic damage which may lead to hepatocellular carcinomas in human and animal livers.     

Identification of aldo-keto reductase AKR1B10 as a selective target for modification and inhibition by prostaglandin A(1): implications for antitumoral activity

Cyclopentenone prostaglandins (cyPG) are reactive eicosanoids that may display anti-inflammatory and antiproliferative actions, possibly offering therapeutic potential. Here we report the identification of members of the aldo-keto reductase (AKR) family as selective targets of the cyPG prostaglandin A(1) (PGA(1)). AKR enzymes metabolize aldehydes and drugs containing carbonyl groups and are involved in inflammation and tumorigenesis. Thus, these enzymes represent a class of targets to develop small molecule inhibitors with therapeutic activity. Molecular modeling studies pointed to the covalent binding of PGA(1) to Cys299, close to the active site of AKR, with His111 and Tyr49, which are highly conserved in the AKR family, playing a role in PGA(1) orientation. Among AKR enzymes, AKR1B10 is considered as a tumor marker and contributes to tumor development and chemoresistance. We validated the direct modification of AKR1B10 by biotinylated PGA(1) (PGA(1)-B) in cells, and confirmed that mutation of Cys299 abolishes PGA(1)-B incorporation, whereas substitution of His111 or Tyr49 reduced the interaction. Modification of AKR1B10 by PGA(1) correlated with loss of enzymatic activity and both effects were increased by depletion of cellular glutathione. Moreover, in lung cancer cells PGA(1) reduced tumorigenic potential and increased accumulation of the AKR substrate doxorubicin, potentiating cell-cycle arrest induced by this chemotherapeutic agent. Our findings define PGA(1) as a new AKR inhibitor and they offer a framework to develop compounds that could counteract cancer chemoresistance.     

Reduced nicotinamide adenine dinucleotide phosphate-dependent formation of 2,3-dihydro-2,3-dihydroxyaflatoxin B1 from aflatoxin B1 by hepatic microsomes.

2,3-Dihydro-2,3-dihydroxyaflatoxin B1 (dihydrodiol) was formed as a major metabolite in the incubation of aflatoxin B1 with rat and hamster liver microsomes. The yield of the dihydrodiol was maximal at pH 6.5, was reduced nicotinmide adenine dinucleotide phosphate- and cytochrome P-450-dependent, and was increased 2- to 4-fold by pretreatment of the animals with phenobarbital; pretreatment with 3-methylcholanthrene did not alter the activity of rat hepatic microsomes. Inhibitors of epoxide hydrase did not lower the yield of the dihydrodiol in these systems. Negligible yields of the dihydrodiol were formed from aflatoxin B1 and rat liver microsomes in the presence of DNA. Little or no formation of the dihydrodiol was noted with microsomes from rat intestinal mucosa, kidney, or lung. These results further support the formation of aflatoxin B1 2,3-oxide as a major electrophilic metabolite of aflatoxin B1 in rat and hamster liver microsomal systems, since this highly reactive epoxide would be expected to hydrolyze readily to form the dihydrodiol.     

Carcinogenicity of aflatoxicol in Fischer 344 rats

Aflatoxicol (AFL), a metabolite of aflatoxin B1 (AFB1), is formed in vitro by liver preparations from several species including humans. A positive correlation appears to exist between the sensitivity of a species to AFB1 and the species ability to metabolize AFB1 to AFL. Conversion of AFB1 to AFL is, therefore, a questionable detoxification step. The carcinogenicity of a diastereoisomeric mixture of AFL, prepared chemically from AFB1, was compared to AFB1 by tumor incidences being determined in 4 groups of 20 weanling male F344 rats fed either a negative control diet with no aflatoxin, a positive 50-ppb AFB1 control diet, a 50-ppb AFL diet, or a 200-ppb AFL diet for 1 year and then killed at the end of the 2d year. The respective hepatocellular carcinoma incidences were 0, 40, 20, and 70%, demonstrating that AFL is carcinogenic in the rat. The data show that a diastereoisomeric mixture of AFL is one-half as carcinogenic as AFB1, and the dose response appeared nearly linear in that a fourfold increase in dose produced a 3.5-fold increase in tumor incidence. The data did not establish unequivocally that AFL is a proximate carcinogen, but metabolism of AFB1 to AFL should not be considered an efficient detoxification reaction.     

Glutathione S-transferase-catalyzed conjugation of bioactivated aflatoxin B(1) in human lung: differential cellular distribution and lack of significance of the GSTM1 genetic polymorphism.

Epidemiological studies suggest that aflatoxin B(1) (AFB(1)), a mycotoxin produced by certain Aspergillus species, may play a role in human respiratory cancers in occupationally-exposed individuals. AFB(1) requires bioactivation to the corresponding exo-8,9-epoxide for carcinogenicity, and glutathione S-transferase (GST)-catalyzed conjugation of the epoxide with glutathione (GSH) is a critical determinant of susceptibility to AFB(1). Of the purified human GST enzymes studied, the polymorphic hGSTM1-1 has the highest activity towards AFB(1) exo-epoxide. The influence of the GSTM1 polymorphism on AFB(1)-GSH formation, as well as the abilities of cytosols from preparations enriched in different isolated lung cell types to conjugate AFB(1)-epoxides, were examined. In whole-lung cytosols from patients undergoing clinically indicated lobectomy, GSTM1 genotype correlated with GSTM1 phenotype as determined by [(3)H]trans-stilbene oxide conjugation: GSTM1-positive = 295 +/- 31 pmol/mg/h (n = 6); GSTM1-negative = 92.8 +/- 23.3 pmol/mg/h (n = 4) (P < 0.05). In contrast, conjugation of microsome-generated [(3)H]AFB(1)-epoxides with GSH was low and variable between patients, and did not correlate with GSTM1 genotype: GSTM1-positive = 11.9 +/- 8.1, 111 +/- 66 and 510 +/- 248 fmol/mg/h (n = 6); GSTM1-negative = 15.3 +/- 16.7, 167 +/- 225 and 540 +/- 618 fmol/mg/h (n = 4) (for 1, 10 and 100 microM [(3)H]AFB(1), respectively). GSH conjugates of AFB(1) exo-epoxide and the much less mutagenic stereoisomer AFB(1) endo-epoxide were produced in a ratio of approximately 1:1 in cytosols from both whole lung and isolated cells. Total cytosolic AFB(1)-epoxide conjugation was significantly higher in fractions enriched in alveolar type II cells (3.07 +/- 1.61 pmol/mg/h) than in unseparated lung cells (0.143 +/- 0.055 pmol/mg/h) or fractions enriched in alveolar macrophages (0. 904 +/- 0.319 pmol/mg/h; n = 4) (P < 0.05). Furthermore, AFB(1)-GSH formation and percentage of alveolar type II cells in different cell fractions were correlated (r = 0.78, P < 0.05). These results demonstrate that human lung GSTs exhibit very low conjugation activity for both AFB(1)-8,9-epoxide stereoisomers, and that this activity is heterogeneously distributed among cell types, with alveolar type II cells exhibiting relatively high activity. Of the GSTs present in human peripheral lung which contribute to AFB(1) exo- and endo-epoxide detoxification, hGSTM1-1 appears to play at most only a minor role.