Exposure to exogenous 17beta-oestradiol disrupts p450aromB mRNA expression in the brain and gonad of adult fathead minnows (Pimephales promelas).

Oestrogens are key regulators in sexual differentiation and development in higher vertebrates. P450 aromatase (p450arom) is the steroidogenic enzyme responsible for the synthesis of oestrogens from aromatisable androgens. Effects of endocrine disrupting chemicals on steroidogenic enzyme gene expression have received little attention so far, yet it is potentially a major pathway for sexual disruption. In this 14-day study the effects of exogenous 17beta-oestradiol (E2) at environmentally relevant concentrations were assessed on gene expression of p450aromB in the gonad and brain of maturing male and female fathead minnows (FHM). Exposure to E2 resulted in an oestrogenic response as shown by a dose-dependent induction of plasma vitellogenin (VTG) in female and male fish and a dose-dependent inhibition of testis growth. There was an effect of exposure to E2 on p450aromB mRNA expression in the gonads; E2 up-regulated p450aromB mRNA expression in the testis and ovary in a dose-response manner after 14 days of exposure. In male brain, p450aromB mRNA concentrations were significantly reduced in fish exposed to 100 and 320 ng E2/l on day 4, but on day 14 were elevated in males exposed to both 32 and 100 ng E2/l. No effects of E2 on p450aromB mRNA expression occurred in the brain of females. The results of this study show that concentrations of E2 found in the environment can have disruptive effects on key steroidogenic enzyme pathways that control sexual development in fish.

Salmonid follicle-stimulating hormone (GtH I) mediates vitellogenic development of oocytes in the rainbow trout, Oncorhynchus mykiss.

Rainbow trout (Oncorhynchus mykiss) were subjected to unilateral ovariectomy (ULO) during early vitellogenesis to examine the endocrine responses mediating the recruitment and growth of oocytes in the secondary (vitellogenic) growth phase. ULO induced recruitment of a second population of primary oocytes into the vitellogenic growth phase that then grew at a faster rate than oocytes in the control fish. Seven days post-ULO, the concentration of plasma salmonid FSH (sFSH = GtH I) was significantly higher than in controls and was elevated for at least 54 days. Maximal concentrations of sFSH in ULO fish (Day 21 post-ULO) were twice (10 ng/ml) those in controls. The data show that sFSH plays a primary role in mediating vitellogenic development. After ULO, plasma concentrations of estradiol-17beta were significantly lower than in controls up until 21 days post-ULO. Thereafter, plasma concentrations of estradiol-17beta did not differ from those in controls. The changes in concentrations of plasma estradiol-17beta and sFSH in the ULO fish demonstrated that the secretion of sFSH is probably not controlled by negative feedback of estradiol-17beta alone; in fish, as in mammals, it is likely that intragonadal autocrine/paracrine factors, such as inhibin and activin, also participate in the regulation of sFSH secretion. Plasma concentrations of testosterone did not appear to differ between the control and ULO fish. The responses in the production of estradiol-17beta and testosterone indicate that the dynamics of sex steroid synthesis in ovarian follicles in ULO fish was different than in the ovaries of control fish.

The effect of oxidative stress on the production of the recombinant protein, interferon gamma, produced by Chinese hamster ovary cells in stirred-batch culture.

The CHO320 cell line, engineered to produce human interferon gamma was investigated with regard to its susceptibility to oxidative stress. Batch cultures of the cells grown in a bench-top bioreactor exhibited no marked response to changes in oxygen concentration between 6% and 14% whereas cell growth and recombinant protein production were inhibited by increasing the oxygen to 20%. High concentrations of hydrogen peroxide (in excess of 200 microM) were required to inhibit growth of the CHO320 cells whereas concentrations of 50 microns and 100 microM had no effect on recombinant protein production. Buthionine sulphoximine (50 microM and 100 microM) completely depleted the cells of glutathione within 24 h; however, no quantitative effect on recombinant protein production was seen. It is concluded that the CHO320 cells are, possibly as a consequence of the long selection process they have undergone, very resistant to oxidative stress.

Scavenging of nitrogen dioxide, thiyl, and sulfonyl free radicals by the nutritional antioxidant beta-carotene.

Mechanisms of free radical scavenging by the nutritional antioxidant beta-carotene have been investigated by pulse radiolysis. Free radicals, which can initiate the chain of lipid peroxidation, including nitrogen dioxide (NO2.), thiyl (RS.), and sulfonyl (RSO2.) radicals, are rapidly scavenged by beta-carotene. Absolute rate constant k[NO2. + beta-carotene] = (1.1 +/- 0.1) x 10(8) m-1 s-1 and for the glutathione thiyl radical k[GS. + beta-carotene] = (2.2 +/- 0.1) x 10(8) m-1 s-1 have been determined. The mechanisms however are mutually exclusive, the former involving electron transfer to generate the radical-cation [ beta-carotene]+. and the latter by radical-addition to generate an adduct-radical [RS... beta-carotene].. Rate constants for thiyl radical-addition reactions vary from 10(6) to 10(9) m-1 s-1 and correlate with the lipophilicity of the thiyl radical under study. Sulfonyl radicals undergo both electron abstraction, [ beta-carotene]+. and radical-addition, [RSO2... beta-carotene]. in an approximate 3:1 ratio. The beta-carotene radical-cation and adduct-radicals are highly resonance stabilized and undergo slow bimolecular decay to non-radical products. These carotenoid-derived radicals react differently with oxygen, a factor which is expected to influence the antioxidant activity of beta-carotene within tissues of varying oxygen tension in vivo.

Comparative evaluation of the antioxidant activity of alpha-tocopherol, alpha-tocopherol polyethylene glycol 1000 succinate and alpha-tocopherol succinate in isolated hepatocytes and liver microsomal suspensions.

The antioxidant activity of alpha-tocopherol polyethylene glycol 1000 succinate (TPGS) and of alpha-tocopherol succinate (TS) has been examined in isolated hepatocytes and microsomal fractions from rat liver. Both TPGS and TS require esterase activity to yield free alpha-tocopherol and, hence, antioxidant activity. TPGS and TS consistently exerted a more effective antioxidant protection than an equivalent amount of directly-added free alpha-tocopherol. The low antioxidant efficiency of directly added free alpha-tocopherol in such water-based experimental systems as used here seems to be due to its extreme hydrophobicity. TPGS, on the other hand, is an extremely hydrophilic compound that is being examined as a useful source of alpha-tocopherol in certain clinical situations and is here shown to be a convenient and effective source for experimental studies into lipid peroxidation and antioxidant mechanisms.

Studies on the hyperplasia ('regeneration') of the rat liver following partial hepatectomy. Changes in lipid peroxidation and general biochemical aspects.

Using the experimental model of partial hepatectomy in the rat, we have examined the relationship between cell division and lipid peroxidation activity. In rats entrained to a regime of 12 h light/12 h dark and with a fixed 8 h feeding period in the dark phase, partial hepatectomy is followed by a rapid regeneration of liver mass with cycles of synchronized cell division at 24 h intervals. The latter phenomenon is indicated in this study by pulses of thymidine kinase activity having maxima at 24 h, 48 h and 72 h after partial hepatectomy. Microsomes prepared from regenerating livers show changes in lipid peroxidation activity (induced by NADPH/ADP/iron or by ascorbate/iron), which is significantly decreased relative to that in microsomes from sham-operated controls, again at 24 h, 48 h and 72 h after the operation. This phenomenon has been investigated with regard to possible underlying changes in the content of microsomal fatty acids, the microsomal enzymes NADPH:cytochrome c reductase and cytochrome P-450, and the physiological microsomal antioxidant alpha-tocopherol. The cycles of decreased lipid peroxidation activity are apparently due, at least in part, to changes in microsomal alpha-tocopherol content that are closely associated in time with thymidine kinase activity.

Studies on lipid peroxidation in normal and tumour tissues. The Yoshida rat liver tumour.

Reduced rates of lipid peroxidation have been observed in Yoshida hepatoma cells and microsomes when compared with appropriate control tissue (normal rat liver) under the same pro-oxidant conditions. The pro-oxidant conditions used were incubation with NADPH+ADP+iron or ascorbate+iron or exposure to gamma-irradiation. As previously shown with the Novikoff hepatoma, the relative concentrations of alpha-tocopherol and polyunsaturated fatty acids are important in conferring resistance to lipid peroxidation in the Yoshida hepatoma. Furthermore, NADPH-cytochrome c reductase and the NADPH-cytochrome P-450 electron transport chain, which are involved in the initiation and propagation of certain types of lipid peroxidation, are found at very much reduced levels in the Yoshida hepatoma. The relative importance of these aberrations are discussed.

Effects of alpha-tocopherol on carbon tetrachloride metabolism in rat liver microsomes.

alpha-tocopherol is the major lipid-soluble radical-scavenging antioxidant in rat liver. It has long been used as a putative protective agent in CCl4 induced liver injury but with variable results. We have used alpha-tocopherol loaded rat liver microsomes to study the effect of this vitamin on CCl4 metabolism in vitro. As expected, alpha-tocopherol inhibits CCl4-dependent microsomal lipid peroxidation and, at a very high concentration, will inhibit the covalent binding of CCl3 to microsomal protein by up to 50%. No inhibitory effect was observed towards CCl3 production as measured by the electron spin resonance technique of spin-trapping but this apparent discrepancy may represent a limitation of the technique. The high levels required to inhibit covalent binding probably preclude the likelihood of alpha-tocopherol significantly affecting that phenomenon at endogenous concentrations but may be relevant to other experiments employing high doses of alpha-tocopherol as an experimental hepatoprotective agent.

Lipid peroxidation in regenerating rat liver.

Rats entrained to a strictly regulated lighting and feeding schedule have been subjected to partial hepatectomy or a sham operation. In the partially hepatectomised animals the period of liver regeneration is characterised by regular bursts of thymidine kinase activity. Liver microsomes from rats, at times corresponding to maximum thymidine kinase activity, have much reduced rates of lipid peroxidation compared to control preparations: this is due in part to increased levels of lipid-soluble antioxidant at times of maximal DNA synthesis. This temporal relationship between thymidine kinase and lipid peroxidation is consistent with the view that lipid peroxidation is decreased prior to cell division.

Low rate of NADPH/ADP-iron dependent lipid peroxidation in hepatic microsomes of DBA/2 mice.

Hepatic microsomal lipid peroxidation has been studied in 4 inbred strains of mice: C57BL/6, BALB/c, AKR and DBA/2. The rates of lipid peroxidation stimulated in vitro by carbon tetrachloride, ascorbate-iron and cumene hydroperoxide were similar in all 4 strains. Lipid peroxidation induced by NADPH/ADP-iron, however, proceeded at a substantially lower rate in the hepatic microsomes of DBA/2 mice. It is suggested that this low rate of enzymic iron-induced lipid peroxidation is a factor that may be involved in the resistance of this strain of mice to experimental hepatic porphyria induced by polyhalogenated aromatic hydrocarbons.