Per Garberg

Studies of the role of DNA fragmentation in selenium toxicity

The role of DNA damage in selenite cytotoxicity was studied in isolated hepatocyte model systems. An initial series of experiments, with hepatocytes in suspension, indicated that selenite-induced DNA fragmentation was oxygen dependent and could be inhibited by cyanide, HgCl2 and CuDIPS. These findings were interpreted to imply that selenite-induced redox cycles were involved in this effect. In a second series of experiments, the effect of inhibitors of poly(ADP-ribose)polymerase (3-aminobenzamide and theophylline) and DNA alkylating agents on selenite-induced cellular lysis was studied. These experiments were performed with hepatocytes in primary culture and 20-30 microM selenite lysed the cultured cells after about 20 hr exposure. It was found that alkylators added 20 hr before selenite acted synergistically with selenite, and that inhibitors of poly(ADP-ribose)polymerase antagonized lysis. Further studies also indicated NAD degradation before lysis. These data indicate a modulating role for DNA damage in selenite cytotoxicity mediated by poly(ADP-ribose)polymerase. Taken together with previously published data on, for example, potentially lethal oxidation of NADPH (Anundi et al., Chem. Biol. Interact. 50, 277, 1984) they also suggest that cell death resulted from interactions between several events that may deplete energy supplies. The results are compatible with a selective killing of DNA-damaged hepatocytes by low doses of selenite.

Recovery of malondialdehyde in urine as a 2,4-dinitrophenylhydrazine derivative analyzed with high-performance liquid chromatography

Malondialdehyde (MDA) in urine was measured as a 2,4-dinitrophenylhydrazine (DNPH) derivative using high-performance liquid chromatography (HPLC) for the analysis. MDA standard coeluted with a peak obtained from rat urine after i.p. administration of MDA standard. This peak was also the only peak containing 14C after injection of a [14C]MDA standard, and was shown by mass spectrometry to contain 1-(2,4-dinitrophenyl)pyrazole, the derivative formed when MDA is treated with DNPH. Depending on the amount given (0.3-5.5 mumol), the recovery (after 24 h sampling period) in urine was 0.7-2.6%. This apparent non-linear kinetics may relate to several factors, such as dose-dependent metabolism. However, the peak urinary concentration approached the expected plasma concentration and reproducible recovery data were obtained, suggesting that MDA was passively excreted in a reasonably stable form. These data indicate that monitoring MDA excretion in urine can give useful information about lipid peroxidation in vivo.

Studies on Se incorporation in selenoproteins; effects of peroxisome proliferators and hydrogen peroxide generating system

The objective of this study was to characterize the influence of peroxisome proliferation on the metabolism of physiological concentrations of Se. In an initial series of experiments hepatocytes in primary cultures and isolated from ordinary-fed rats, were used. The cells were exposed to 75Se-selenite (30 nM) and after 24 h the labelling of selenoproteins was analysed with SDS-PAGE. Treatments with mono(2-ethylhexyl)phthalate (MEHP; a metabolite of di(2-ethylhexyl)phthalate (DEHP)), nafenopin, decreased oxygen tension and a H2O2 generating system decreased the labelling of a 23-kDa and a 15-kDa protein. The decreased labelling of the 23- and the 15-kDa proteins was usually accompanied by an increased labelling of a 58-kDa protein. Increased oxygen tension induced uncertain effects, possibly due to toxicity. In order to further evaluate the validity of the model, the labelling was also studied in hepatocytes isolated from Se-deficient and torula yeast-fed rats. In these cells there was a decreased labelling of the 23-kDa protein as compared to cells from Se-supplemented controls when 100 nM selenite was used. In in vivo experiments it was found that a DEHP-induced decrease in glutathione peroxidase (GSH-Px) activity was potentiated by high doses of selenite. To a large extent, the labelling data are compatible with enzyme activity data and in vivo data. For example, the decreased labelling of the 23-kDa protein may reflect the decreased GSH-Px activity. It is concluded that the effects induced by MEHP on Se-labelling can be explained by an increase in the steady state level of H2O2.

Decreased glutathione peroxidase activity in mice in response to nafenopin is caused by changes in selenium metabolism.

The activity of selenium-dependent glutathione peroxidase is known to be reduced in the liver of both rats and mice after exposure to nafenopin, as well as other peroxisome proliferators. The mechanism for this down-regulation is not known, but might involve changes in incorporation of selenium into selenoproteins. In this paper we show that both incorporation of selenium into selenoproteins and the level of selenium in liver is reduced in mice treated with nafenopin. The activity of selenium dependent glutathione peroxidase (GPx), as well as incorporation of selenium into its 23 kD subunit were found to be decreased. Contrary to what might have been expected, the decreased GPx activity was detected concomitantly with a slight increase in mRNA levels after 10 days of treatment, while a small decrease in mRNA levels was detected in treated animals after 26 weeks, together with the decrease in GPx-activity. Incorporation of selenium into liver fatty acid binding protein (L-FABP) was also decreased, even though large increases in protein and mRNA levels were detected. Taken together these data suggest that the decrease in GPx-activity in response to nafenopin is due to post-transcriptional mechanisms, involving changes in selenium metabolism.

The involvement of selenium in peroxisome proliferation caused by dietary administration of clofibrate to rats

The effects of dietary treatment with clofibrate (0.5% w/w for 10 days) on the livers of selenium-deficient male rats were examined. The peroxisome proliferation (as determined by electron microscopy) in the livers of selenium-deficient animals was much less pronounced than in the case of selenium-adequate rats and no increase in peroxisomal fatty acid beta-oxidation (assayed both as antimycin-insensitive palmitoyl-CoA oxidation and lauroyl-CoA oxidase activity) was observed in the deficient animals. On the other hand, in selenium-deficient rats clofibrate caused increases in the specific activity of microsomal lauric acid omega- and omega-1-hydroxylation and an apparent change in mitochondrial size, seen as a redistribution of mitochondria from the 600 x g(av) pellet to the 10,000 x g(av) pellet, which were approximately 50% as great as the corresponding effects on control animals. Obviously, then, these three different effects of clofibrate are not strictly coupled and may involve at least partially distinct underlying mechanisms. Initial experiments demonstrated that peroxisome proliferation could be obtained by exposing primary hepatocyte cultures derived from selenium-deficient rats to clofibric acid (an in vivo hydrolysis product of clofibrate which is the proximate peroxisome proliferator), nafenopin or mono(2-ethylhexyl)phthalate. This finding suggests that selenium deficiency does not have a direct influence on the basic process(es) underlying peroxisome proliferation, but rather has indirect effects, influencing, for example, the pharmacokinetics of clofibrate and/or hormonal factors.

Studies on Selenite-Induced DNA Fragmentation and the Role of Poly(ADP Ribose)Polymerase in Selenite Toxicity

Selenium has been shown to act as an anticarcinogen in many experimental studies but it is also a potent toxin, and the question may be raised whether anticarcinogenic effects can be mediated by cytotoxicity. It is, for example, known that one of the toxic effects of selenium is DNA fragmentation (Shamberger 1985), and DNA fragmentation induced by other toxic substances can lead to an activation of the nuclear enzyme poly (ADP-ribose)polymerase (PARP) (Berger 1985). This enzyme utilizes NAD as a substrate, and it has been proposed that an extensive DNA fragmentation may lead to cell death via NAD depletion. It is thus possible that selenite can interact with other DNA-damaging compounds so that it selectively kills DNA-damaged cells.