James L. Gardner

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.

Newly described form of X-linked arthrogryposis maps to the long arm of the human X chromosome

Arthrogryposis is a heterogeneous birth defect characterized by limitation of movement at multiple joints. One in 3,000 infants is born with arthrogryposis, and at least a third of these cases have a genetic cause. Four distinct types of X-linked arthrogryposis have been reported, and a severe lethal form recently was mapped to Xpll.3-qll.2. We now report an extended family affected with a novel variant of X-linked arthrogryposis that involves only the lower limbs. Linkage analysis with polymorphic DNA markers maps the disease locus in this unique family to the long arm of the human X chromosome between DXS1220 and DXS1205 in Xq23-27.

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.

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.