Pyruvate inhibits clofibrate-induced hepatic peroxisomal proliferation and free radical production in rats.

In an effort to identify the effects of the 3-carbon compound pyruvate on free radical production, we measured hepatic total peroxisomal beta-oxidation and catalase activity and the production of lipofuscin-like products in male Sprague-Dawley rats consuming an adequate diet supplemented with pyruvate, vitamin E, or the peroxisome proliferator and free radical enhancer clofibrate for 22 days (n = 5 in each group). Clofibrate feeding induced hepatomegaly, a fivefold increase in total peroxisomal beta-oxidation activity, and a threefold increase in hepatic lipofuscin-like products (P < .05). Pyruvate but not vitamin E inhibited the increase in liver size by 70% (P < .05). Both pyruvate and vitamin E completely inhibited clofibrate-induced increases in lipofuscin-like products (P < .05). Pyruvate but not clofibrate or vitamin E increased plasma concentrations of the nitric oxide metabolites nitrite and nitrate (P < .05). We conclude that with clofibrate-induced peroxisomal proliferation and free radical production, pyruvate will inhibit peroxisomal proliferation and free radical production, inhibit free radical-induced lipid peroxidation, and enhance metabolism of nitric oxide.

The relevance of hepatic peroxisome proliferation in rats to assessment of human carcinogenic risk for pharmaceuticals.

There exists a strong association between the ability of drugs and other chemicals to induce hepatic peroxisomes in rodents and to increase the frequency of liver tumors in these same species. Despite several years of intensive investigation, a plausible mechanism causally relating peroxisome induction to tumor formation has not been found. Two major theories of how such compounds produce tumors--prolonged oxidative stress and cellular growth dysregulation--have limited experimental support. The oxidative stress demonstrated in rodents as a consequence of peroxisomal activity may be irrelevant to man since primates appear to be much less susceptible to peroxisome proliferation than are rats or mice. Cellular growth dysregulation and other effects of test materials that often accompany--but are not directly attributable to--peroxisome proliferation in rodents can be assessed in other, more relevant species if a causal relationship between their presence and tumor development is ultimately shown in rodents. Currently, there is no reason to specifically avoid the clinical assessment and commercial development of new therapeutic drugs that induce peroxisomes in rodents if other measurements indicate the absence of DNA damage or growth dysregulation at reasonable exposures in relevant species.

Further studies on the involvement of selenium in peroxisome proliferation in rat liver. Comparison of effects with clofibric acid and perfluorooctanoic acid and the pharmacokinetics of [14C]clofibrate.

Most effects of the peroxisome proliferator clofibrate on rat liver are marginal or absent in selenium (Se) deficiency. The purpose of the present study was to determine whether the uptake or distribution of clofibrate is altered by Se deficiency. Rats were fed a Se-adequate or -deficient diet for 10-11 weeks and then these same diets with 0.5% (w/w) clofibric acid (the direct acting hydrolysis product of clofibrate) or 0.02% (w/w) perfluorooctanoic acid (PFOA) for 10 days. Other groups of rats received radiolabeled clofibrate by intubation. Clofibric acid was an ineffective as clofibrate in producing effects (i.e. decreased body weight gain, increases in liver somatic index and protein content of the mitochondrial fraction, and increased activities of catalase and peroxisomal fatty acid beta-oxidation) in the liver of Se-deficient rats. Microsomal omega-hydroxylation was, however, equally induced in both dietary groups. In contrast to clofibric acid, the biological effects of PFOA were not affected by Se status. Furthermore, neither the tissue distribution (plasma, liver and kidney) nor the urinary excretion of 14C was affected by Se deficiency. These results demonstrate that the hydrolysis of clofibrate to clofibric acid is not impaired in the Se-deficient rat. In addition, the involvement of Se in the effects of peroxisome proliferators differs for different members of this structurally heterogeneous group of compounds. It is concluded that the Se-deficient rat may provide valuable information concerning the biochemical mechanism(s) underlying peroxisome proliferation.

Expression of antisense nodulin-35 RNA in Vigna aconitifolia transgenic root nodules retards peroxisome development and affects nitrogen availability to the plant.

A nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3.) was isolated from a Vigna aconitifolia (mothbean) root nodule cDNA library. Sequence analysis of Vigna uricase (VN-35) cDNA revealed 90% homology to that of soybean. The VN-35 cDNA was inserted in the antisense orientation downstream of the caMV-35S promoter, and transgenic hairy roots were formed on Vigna plants using Agrobacterium rhizogenes. Infection with Bradyrhizobium (cowpea) gave rise to root nodules on transgenic hairy roots supported by the wild-type shoot. Expression of antisense VN-35 RNA was detected in transgenic nodules on individual roots using polymerase chain reaction (PCR). The nodules expressing antisense VN-35 RNA were smaller in size and showed lower uricase activity than nodules formed on the hairy roots transformed with a binary vector containing beta-glucuronidase (GUS) gene (used as control), and the plants exhibited nitrogen deficiency symptoms. Ultrastructural analysis and immunogold labeling with antibody against soybean N-35 revealed that the growth of peroxisomes was retarded in transgenic nodules expressing antisense VN-35 RNA. These data suggest that a reduction in ureide biosynthesis limits the availability of symbiotically reduced nitrogen to the plant. The nodules of tropical legumes appear to be specialized in nitrogen assimilation and are developmentally controlled to produce and transport ureides.

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.