Glutathione transferases (GSTs) of a novel class, which it is proposed to term Theta, were purified from rat and human liver. Two, named GST 5-5 and GST 12-12, were obtained from the rat, and one, named GST theta, was from the human. Unlike other mammalian GSTs they lack activity towards 1-chloro-2,4-dinitrobenzene and are not retained by GSH affinity matrices. Only GST 5-5 retains full activity during purification, and its activities towards the substrates 1,2-epoxy-3-(p-nitrophenoxy)propane, p-nitrobenzyl chloride, p-nitrophenethyl bromide, cumene hydroperoxide, dichloromethane and DNA hydroperoxide are 185, 86, 67, 42, 11 and 0.03 mumol/min per mg of protein respectively. Earlier preparations of GST 5-5 or GST E were probably a mixture of GST 5-5 and GST 12-12, which was largely inactive, and may also have been contaminated by less than 1% with another GSH peroxidase of far greater activity. Partial analysis of primary structure shows that subunits 5, 12 and theta are related to each other, particularly at the N-terminus, where 25 of 27 residues are identical, but have little relationship to the Alpha, Mu and Pi classes of mammalian GSTs. They do, however, show some relatedness to subunit I of Drosophila melanogaster [Toung, Hsieh & Tu (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 31-35] and the dichloromethane dehalogenase of Methylobacterium DM4 [La Roche & Leisinger (1990) J. Bacteriol, 172, 164-171].

The ontogeny of rat liver glutathione S-transferase (EC 2.5.1.18) (GSTs) during foetal and postnatal development was investigated. The GSTs are dimers, the subunits of which belong to three multigene families, Alpha (subunits 1, 2, 8 and 10), Mu (subunits 3, 4, 6, 9 and 11) and Pi (subunit 7) [Mannervik, Alin, Guthenberg, Jennsson, Tahir, Warholm & Jörnvall (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7202-7206; Kispert, Meyer, Lalor, Coles & Ketterer (1989) Biochem. J. 260, 789-793]. There is considerable structural homology within each gene family, with the result that whereas reverse-phase h.p.l.c. successfully differentiates individual subunits, immunocytochemical and Northern-blotting analyses may only differentiate families. Enzymic activity, h.p.l.c. and Northern blotting indicated that expression of GST increased from very low levels at 12 days of foetal growth to substantial amounts at day 21. At birth, GST concentrations underwent a dramatic decline and remained low until 5-10 days post partum, after which they increased to adult levels. During the period under study, GST subunits underwent differential expression. The Mu family had a lower level of expression than the Alpha family, and, within the Alpha family, subunit 1 was more dominant in the adult than the foetus. Subunit 2 is the major form in the foetus. Most noteworthy were subunits 7 and 10, which were prominent in the foetus, but present at low levels post partum. Immunocytochemical analysis of the 17-day foetal and newborn rat livers showed marked differences in the distribution of GSTs in hepatocytes. In the 17-day foetal liver Pi greater than Alpha greater than Mu whereas in the newborns Alpha greater than Mu much greater than Pi. Erythropoietic cells were not stained for any of the three GST families. Steady-state mRNA concentrations in the foetus correlated with the relative transcription of the Alpha, Mu and Pi class genes. However, in those genes expressed post partum, namely the Alpha and Mu class, low transcriptional activity was associated with high concentrations of mRNA. This suggests that there is a switch from transcriptional control to post-transcriptional control at birth. GST 7-7 appears to be regulated predominantly by transcription throughout the period of liver development under observation.

Summary The spontaneous reaction of 110 ILM chlorambucil (4-[p-[bis(2-chloroethyl) However, GTSs Al-I and A2-2 were associated with a significant increase of CHBSG at the expense of CHBSG2 + CHBSGOH suggesting that these GTSs sequestered CHBSG at the active site. This interpretation was supported by inhibition studies which showed that CHBSG was a pure competitive inhibitor of the activity of GSTs Al-l and A2-2 towards 1-chloro-2,4-dinitrobenzene with Ki's of 1.3 and 1.2 tiM respectively. GSH transferases P1-1 and Mla-la were inhibited by CHBSG above 10 LM.Incubation of 2 tLM CHB, a concentration which may be of more significance for chemotherapy, in the presence or absence of GST A 1-2 (20-50 LM) showed catalysis of GSH monoconjugation equivalent to 18% of the spontaneous rate. However, the dominant effect again was the sequestration of CHBSG which reached 74.3 ± 1.5 (SEM)% of the total reactants at 60 min compared to 28.9 ± 0.3(SEM)% in controls. CHBSG, although possessing a potential electrophilic centre, showed no detectable alkylation of plasmid DNA but indirect evidence was obtained that it alkylated other cellular macromolecules.It is concluded that the contribution of GSTs to catalysis of CHB detoxication will depend on factors not previously considered, namely the relative molarities of CHB, CHBSG and GSTs, and the cellular capacity to excrete CHBSG to relieve product inhibition. CHB is a hydrophobic anion at physiological pH and was shown by Bank et al. (1989) to enter and leave leukaemic lymphocytes rapidly by simple diffusion. In plasma, CHB is stabilised by binding to albumin (Ehrsson et al., 1981).A major problem in the use of cancer chemotherapeutics is the development of cellular resistance and, in the case of alkylating agents such as CHB, numerous studies suggest that enhanced expression of glutathione transferases (GSTs) may contribute to such resistance. For instance: Lewis et al. (1988) found that amplification of an alpha class GST in a Chinese hamster ovary (CHO) cell line was associated with resistance to CHB and other nitrogen mustards; Puchalski and Fahl (1990) found increased resistance to CHB upon transfection of mouse and monkey cells with rat GSTs 1-1 (class alpha), 3-3 (class mu) or human GST P1-1 (class pi, for nomenclature of human GSTs see Mannervik et al., 1992), and Johnston et al. (1990) found an inverse correlation between both GSH and total GST activity and CHB-dependent DNA adducts formed in vitro in human chronic lymphocytic leukaemic lymphocytes. In support of these observations, murine GSTs of alpha, mu and pi classes were shown by Ciaccio et al. (1990) to increase significantly the rate of formation of CHBSG in vitro at pH 6.5, and the alpha class enzyme also stimulated the second GSH conjugation. Recently, Ciaccio et al. (1991) have also observed catalysis of the GSH conjugaton of CHB by human GSTs of the alpha and pi classes. In contrast, the transfection of GST P1-I into NIH 3T3 cells failed to alter their sensitivity to CHB (Nakagawa et al., 1990). Moreover, L...

Male and female Sprague--Dawley rats were maintained for 5 days on control diet or diet containing 0.075% (w/w) 1,2-dithiole-3-thione (D3T) or 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz). The content of GSH transferase (GST) subunits 1a, 1b, 2, 3, 4, 6, 7, 8, 10 and 11 in the soluble fraction of liver, kidney, small intestine, stomach and lung of control and D3T-fed animals was determined. Liver and kidney were similarly analysed for the oltipraz-fed animals. Significant induction of GST subunits by D3T was seen in all tissues, the most substantial being subunit 7 in male liver (approximately 50-fold) and subunits 1b, 3 and 10 in male and female small intestine (5- to 16-fold). Generally subunits 7, 10, 1b and 3 were most affected, while subunits 6 and 8 were hardly inducible. Oltipraz caused qualitatively similar inductions in the liver and kidney. The effect of GSH transferase induction on the hepatic capacity for GSH conjugation of aflatoxin B1 exo-8,9-oxide is assessed.

The constitutive and inducible cytosolic glutathione S-transferase (EC 2.5.1.18) subunit compositions of parenchymal cells (hepatocytes) and biliary epithelial cells (BEC) from rat liver have been quantitatively analysed using reverse-phase h.p.l.c. Hepatocytes, analysed in the absence of non-parenchymal cells, expressed constitutively the following subunits, in order of their concentration: 3, 4, 2, 1a, 1b, 8, 6 and 10. BEC express constitutively only four of the GST subunits expressed by hepatocytes and these are, in order of their concentration: subunits 2, 7, 4 and 3. Notable differences from hepatocytes are that BEC completely lack the Alpha-class subunits 1a and 1b that are major subunits in hepatocytes, Mu-class subunits make up a very low proportion of the total, and the Pi-class subunit 7 is a major subunit in BEC, whereas it is essentially absent from hepatocytes. For the first time, the effects of the inducing agents phenobarbitone (PB), beta-naphthoflavone (beta-NF) and ethoxyquin (EQ) have been characterized in a comprehensive and quantitative manner in both cell types. PB, beta-NF and EQ increased total GST protein in hepatocytes by approx. 2-fold, 3-fold and 4-fold respectively. Subunits significantly induced in hepatocytes were (in order of fold-induction): by PB, 1b > 8 > 3 > 2 > 4; by beta-NF, 1b > 8 > 2 > 3 > 4; and by EQ, 7 > 1b > 10 > 8 > 3 > 2 > 1a > 4. In BEC, neither PB nor beta-NF had significant effects on the total amount of GST protein, although PB did significantly induce subunit 3 at the expense of other subunits. EQ increased total GST protein nearly 5-fold in BEC, subunits 7 and 3 being induced dramatically above constitutive levels.