Evan P. Gallagher

Decreased glutathione S-transferase expression and activity and altered sex steroids in Lake Apopka brown bullheads (Ameiurus nebulosus).

A number of freshwater lakes and reclaimed agricultural sites in Central Florida have been the receiving waters for agrochemical and municipal runoff. One of these sites, Lake Apopka, is also a eutrophic system that has been the focus of several case studies reporting altered reproductive activity linked to bioaccumulation of persistent organochlorine chemicals in aquatic species. The present study was initiated to determine if brown bullheads (Ameiurus nebulosus) from the north marsh of Lake Apopka (Lake Apopka Marsh) exhibit an altered capacity to detoxify environmental chemicals through hepatic glutathione S-transferase (GST)-mediated conjugation as compared with bullheads from a nearby reference site (Lake Woodruff). We also compared plasma sex hormone concentrations (testosterone, 17-beta estradiol, and 11 keto-testosterone) in bullheads from the two sites. Female bullheads from Lake Apopka had 40% lower initial rate GST conjugative activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 50% lower activity towards p-nitrobutyl chloride (NBC), 33% lower activity toward ethacrynic acid (ECA), and 43% lower activity toward Delta5-androstene-3,17-dione (Delta(5)-ADI), as compared with female bullheads from Lake Woodruff. Enzyme kinetic analyses demonstrated that female bullheads from Lake Apopka had lower GST-catalyzed CDNB clearance than did female Lake Woodruff bullheads. Western blotting studies of bullhead liver cytosolic proteins demonstrated that the reduced GST catalytic activities in female Lake Apopka bullheads were accompanied by lower expression of hepatic GST protein. No site differences were observed with respect to GST activities or GST protein expression in male bullheads. Female Lake Apopka bullheads also had elevated concentrations of plasma androgens (testosterone and 11-ketotestosterone) as compared with females from Lake Woodruff. In contrast, male Lake Apopka bullheads had elevated levels of plasma estrogen but similar levels of androgens as compared with male bullheads from Lake Woodruff. Collectively, our studies indicate the presence of reduced GST protein expression, reduced GST conjugative capacity and altered sex steroid homeostasis in female bullheads from a contaminated field site in Central Florida. The implications of these physiological alterations in terms of pollutant biotransformation and reproduction are discussed.

Comparative expression of two alpha class glutathione S-transferases in human adult and prenatal liver tissues.

The ability of the fetus to detoxify transplacental drugs and chemicals can be a critical determinant of teratogenesis and developmental toxicity. Developmentally regulated expression of alpha class glutathione S-transferases (GSTs) is of particular interest, since these isozymes have high activity toward peroxidative byproducts of oxidative injury that are linked to teratogenesis. The present study was initiated to examine the expression and catalytic activities of alpha class GST isozymes in human prenatal liver. Northern analysis demonstrated the presence of hGSTA1 and/or A2 (hGSTA1/2) and hGSTA4 steady-state mRNAs in second trimester prenatal livers. Western blotting of prenatal liver proteins provided corroborating evidence via detection of an hGSTA1/2-reactive protein in both cytosol and mitochondria and of hGSTA4-4-reactive protein in mitochondria alone. Catalytic studies demonstrated that prenatal liver cytosolic GSTs were active toward 1-chloro-2,4-dinitrobenzene (a general GST reference substrate), delta5-androstene-3,17-dione (relatively specific for hGSTA1-1), and 4-hydroxynonenal, a highly mutagenic alpha,beta-unsaturated aldehyde produced during oxidative damage and a substrate for hGSTA4-4. Total GSH-peroxidase and GST-dependent peroxidase activities were 9- and 18-fold higher, respectively, in adult liver than in prenatal liver. Multiple tissue array analyses demonstrated considerable tissue-specific and developmental variation in GST mRNA expression. In summary, our results demonstrate the presence of two important alpha class GSTs in second trimester human prenatal tissues, and indicate that mitochondrial targeting of GST may represent an important pathway for removal of cytotoxic products in prenatal liver. Furthermore, the relatively inefficient prenatal reduction of hydroperoxides may underlie an increased susceptibility to maternally transferred pro-oxidant drugs and chemicals.

Altered glutathione S-transferase catalytic activities in female brown bullheads from a contaminated central Florida lake

Brown bullheads (Ameriurus nebulosus) are a demersal freshwater species that can be found in a number of polluted ecosystems. The purpose of the present study was to determine the overall capacity for in vitro glutathione S-transferase (GST) detoxification by brown bullheads, and to see if bullhead GST catalysis was altered in bullheads from a polluted site. Brown bullhead liver cytosolic GSTs catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) over a large range of substrate concentrations, with apparent Km and Vmax for CDNB at fixed nucleophile (glutathione, GSH) concentrations of 1.8-1.9 mM and 12.1-14.6 mumol CDNB conjugated/min/mg, respectively. Bullhead GSTs were also highly active toward other substrates such as ethacrynic acid (ECA), delta 5-androstene-3,17-dione (ADI), and nitrobutyl chloride (NBC). Initial rate GST catalytic activities toward CDNB, NBC, ECA, and ADI were significantly lower in female bullheads from a contaminated lake (Lake Apopka Marsh) as compared to female bullheads inhabiting a nearby control site (Lake Woodruff). No site differences were observed with respect to male bullhead GST activities. These studies suggest that brown bullheads efficiently carry out GST conjugation of diverse electrophilic substrates. However, bullhead GST catalysis may be compromised in bullheads inhabiting polluted ecosystems.

Isolation and cloning of homologous glutathione S-transferase cDNAs from English sole and starry flounder liver

Abstract We are currently investigating hepatic glutathione S-transferase (GST) expression in English sole (Pleuronectes vetulus) and starry flounder (Platichthys stellatus), two closely related benthic fish that exhibit striking differences in the prevalences of contaminant-associated liver neoplasia. Alignment of cDNA sequences encoding plaice (Pleuronectes platessa) liver GST (PLGST-A, a theta class GST, Leaver et al., 1993 ) and a GST-related lens crystallins protein from octopus (Octopus vulgaris) revealed conserved regions for oligonucleotide primer design for polymerase chain reaction (PCR)-based studies of sole and flounder GSTs. Reverse-transcriptase-PCR (RT-PCR) analysis of sole and flounder hepatic total mRNA yielded 469 nucleotide cDNA fragments that displayed extensive sequence homology (98%) to plaice GST-A. Northern blotting analysis of English sole and starry flounder liver using the PCR product from English sole as a probe detected a single band of approximately 1000 nucleotides that was highly expressed in English sole and in starry flounder liver. Biochemical and immunological studies using diagnostic GST reference substrates and class specific polyclonal antibodies suggested little homology among English sole and starry flounder GSTs, and rodent alpha, mu, and pi class GSTs. Interestingly, English sole, the species exhibiting a relatively high prevalence of contaminant-associated liver neoplasia, exhibited higher hepatic GST activity toward the environmental epoxides aflatoxin B1-8,9-epoxide (AFBO) and benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide-anti (BPDE). BPDE, in particular, is a carcinogenic epoxide intermediate implicated as a possible causative agent in the etiology of these hepatic lesions. In summary, our studies indicate that a GST related to non-mammalian theta class GST is conserved in marine flatfish. Further mechanistic studies are necessary to determine the role of GSTs in the observed susceptibility differences among English sole and starry flounder to sediment-associated carcinogens.

Conjugation of 4-hydroxynonenal by largemouth bass (Micropterus salmoides) glutathione S-transferases.

The glutathione S-transferases (GST) are a major group of conjugative enzymes involved in the detoxification of electrophilic compounds and products of oxidative stress. We have previously described the kinetics of hepatic GST conjugation in largemouth bass using a variety of synthetic GST reference substrates. In the present study, we investigated the ability of largemouth bass hepatic GSTs to conjugate 4-hydroxynon-2-enal (4HNE), a mutagenic and cytotoxic alpha-beta-unsaturated aldehyde produced during oxidative injury. Hepatic cytosolic fractions from largemouth bass rapidly catalyzed GSH-dependent 4HNE conjugation, with the rate of GST-4HNE conjugation in bass liver exceeding those of several other mammalian and aquatic species. No apparent sex-related differences in GST-4HNE activity were observed among adult bass. SDS-PAGE and Western blotting analysis of GSH affinity-purified bass liver cytosolic GST revealed the presence of two major GST subunits of approximately 30 and 27 KDa that exhibited slight cross-reactivity when probed with a rat alpha class GST antibody, but not to rat mu, pi or theta class GST. The rapid conjugation of 4HNE by hepatic GST suggests an important role for GSTs in protecting against peroxidation of polyunsaturated fatty acids in bass liver.

Conservation of a glutathione S-transferase in marine and freshwater fish.

We have previously reported the isolation and cloning of glutathione S-transferase (GST) cDNAs from two marine fish, English sole (Pleuronectes vetulus) and starry flounder (Platichthys stellatus), that exhibited > 95% identity to plaice (Pleuronectes platessa) GST-A Aquatic Toxicol., 44, 171-182]. In the present study, we have used reverse transcription-polymerase chain reaction (RT-PCR) analysis to isolate a 471 nucleotide GST-like cDNA from largemouth bass (Micropterus salmoides) liver. Sequence identity of the largemouth bass partial cDNA to plaice GST-A was approximately 90%. Northern blotting analysis using the partial GST cDNA from English sole as a probe detected a single band of approximately 1 kb in English sole liver and a slightly larger GST-like band that was highly expressed in largemouth bass liver. In addition, a faint band of similar size was recognized in brown bullhead (Ameriurus nebulosus) liver, but not in channel catfish (Ictalurus punctatus) liver. In conclusion, we have extended our studies of GST expression in flatfish and have isolated an additional GST-A-like cDNA from a largemouth bass. Conservation of a GST-A like cDNA among certain marine flatfish and freshwater species suggests an important function for this gene.

Ontogenic differences in human liver 4-hydroxynonenal detoxification are associated with in vitro injury to fetal hematopoietic stem cells

4-hydroxynonenal (4HNE) is a highly mutagenic and cytotoxic alpha,beta-unsaturated aldehyde that can be produced in utero during transplacental exposure to prooxidant compounds. Cellular protection against 4HNE injury is provided by alcohol dehydrogenases (ADH), aldehyde reductases (ALRD), aldehyde dehydrogenases (ALDH), and glutathione S-transferases (GST). In the present study, we examined the comparative detoxification of 4HNE by aldehyde-metabolizing enzymes in a panel of adult and second-trimester prenatal liver tissues and report the toxicological ramifications of ontogenic 4HNE detoxification in vitro. The initial rates of 4HNE oxidation and reduction were two- to fivefold lower in prenatal liver subcellular fractions as compared to adult liver, and the rates of GST conjugation of 4HNE were not detectable in either prenatal or adult cytosolic fractions. GSH-affinity purification of hepatic cytosol yielded detectable and roughly equivalent rates of GST-4HNE conjugation for the two age groups. Consistent with the inefficient oxidative and reductive metabolism of 4HNE in prenatal liver, cytosolic fractions prepared from prenatal liver exhibited a decreased ability to protect against 4HNE-protein adduct formation relative to adults. Prenatal liver hematopoietic stem cells (HSC), which constitute a significant percentage of prenatal liver cell populations, exhibited ALDH activities toward 4HNE, but little reductive or conjugative capacity toward 4HNE through ALRD, ADH, and GST. Cultured HSC exposed to 5 microM 4HNE exhibited a loss in viability and readily formed one or more high molecular weight 4HNE-protein adduct(s). Collectively, our results indicate that second trimester prenatal liver has a lower ability to detoxify 4HNE relative to adults, and that the inefficient detoxification of 4HNE underlies an increased susceptibility to 4HNE injury in sensitive prenatal hepatic cell targets.