Effect of simultaneous exposure to lead and cadmium on gonadotropin binding and steroidogenesis on granulosa cells: an in vitro study.

Effects of lead (Pb) and cadmium (Cd) both alone or in combination on the binding of LH and FSH on isolated granulosa cells were studied. Granulosa cells isolated from proestrous rats were incubated (in vitro) with lead acetate and/or cadmium acetate (0.03 microM of Pb or Cd) for 1 hr. LH binding was dropped to 84% in Pb treated cells, 72.5% in Cd treated cells and 74.8% in combined metal treated cells compared to control. FSH binding dropped to 85.5% in Pb treated cells, 71.16% in Cd treated cells and 72.5% in combined metal treated cells compared to control. Activity of 17beta Hydroxy Steroid Dehydrogenase (17betaHSDH), a key steroidogenic enzyme was reduced by 52% in Cd and 37% in combined metal exposed cells whereas Pb exposed cells showed 31% reduction in the enzyme activity. Pretreatment with SH groups protectants (glutathione [GSH], dithiothretol [DTT]) and zinc caused an ameriolation in enzyme activity whereas Zn pretreatment showed an increase in gonadotropin binding in metal exposed cells. These results suggest that both Pb and Cd can cause a reduction in LH and FSH binding, which significantly alters steroid production in vitro and exerts a direct influence on granulosa cell function.

Effects of lead and/or zinc exposure during the second stage of rapid postnatal brain growth on delta-aminolevulinate dehydratase and negative geotaxis of suckling rats

Lead has been shown to produce cognitive and motor deficits in young rats that could be mediated, at least in part, by inhibition of the zinc-containing heme biosynthetic enzyme delta-aminolevulinate dehydratase (ALA-D). In the present study we investigated the effects of lead and/or zinc treatment during the second stage of rapid postnatal brain development on brain, kidney and blood ALA-D specific activity, as well as the negative geotaxis behavior of rats. Eight-day-old Wistar rats were injected intraperitoneally with saline, lead acetate (8 mg/kg) and/or zinc chloride (2 mg/kg) daily for five consecutive days. Twenty-four hours after treatment, ALA-D activity was determined in the absence and presence of DL-dithiothreitol (DTT). The negative geotaxis behavior was assessed in 9- to 13-day-old rats. Treatment with lead and/or zinc did not affect body, brain or kidney weights or brain- or kidney-to-body weight ratios of the animals. In spite of the absence of effect of any treatment on ALA-D specific activity in brain, kidney and blood, the reactivation index with DTT was higher in the groups treated with lead or lead + zinc than in the control group, in brain, kidney and blood (mean + or - SEM; brain: 33.33 + or - 4.34, 38.90 + or - 8.24, 13.67 + or - 3.41; kidney: 33.50 + or - 2.97, 37.60 + or - 2.67, 15.80 + or - 2.66; blood: 63.95 + or - 3.73, 56.43 + or - 5.93, 31.07 + or - 4.61, respectively, N = 9-11). The negative geotaxis response behavior was not affected by lead and/or zinc treatment. The results indicate that lead and/or zinc treatment during the second stage of rapid postnatal brain growth affected ALA-D, but zinc was not sufficient to protect the enzyme from the effects of lead in brain, kidney and blood.

Rat epididymal sperm exhibit on dithiothreitol treatment in vitro quantifiable differences in patterns of light scatter, uptake of 14C-iodoacetamide and binding of ethidium bromide to DNA.

The extent to which chromatin of rat caput (CAP), corpus (COR), cauda (CAU) spermatozoa undergo condensation and compaction is known to be a function of progressive increase in the formation of inter- as well as intra-protamine disulphide bridges during their transit through the epididymis. Relative compaction undergone by the nuclear chromatin of these sperm populations was studied by monitoring their susceptibility to in vitro decondensation induced by varying concentrations (0, 0.01, 1, 5, 10, 50 mM) of disulphide reducing agent, dithiothreitol (DTT) after an initial exposure to 0.01% papain. Following this treatment and staining with the nucleic acid specific fluorochrome, ethidium bromide (EB), it was observed that irrespective of the epididymal region from which they were collected, spermatozoa exhibited DTT dose-dependent (a) increase in nuclear size as seen under fluorescence microscopic examination, (b) decrease in flow cytometrically quantifiable light scatter parameters--forward scatter (FSc, 'nuclear size') and side scatter (SSc, nuclear 'granularity'), (c) increase in individual cell EB binding when analyzed by DNA flow cytometry, and (d) increase in thiol specific 14C-iodoacetamide (14C-IA) uptake. The decrease in both FSc and SSc occurring in spite of actual increase in nuclear size has been attributed to increase in translucency of spermatozoan nuclei consequent to decondensation. The FSc, SSc and EB bindability were studied by monitoring both the channels of maximal cell concentration detected in the flow cytograms as well as by digitally quantitating the numbers of cells within specific channels (1-64, 65-128, 129-192 and 193-256) of the flow cytogram. The latter indicated a measure of the variability in the response of populations of sperm within each sample to DTT induced decondensation. At any given concentration of DTT, especially between 5-10 mM, the differences observed between sperms of different regions were consistent and significant (P < 0.01-P < 0.001), maximal changes being shown by CAP and minimal by CAU sperm, COR sperm appearing in between. The effective concentration of DTT required to elicit 50% of maximal (i.e. that exhibited by CAP sperm when taken as 100%) effect (ED50) varied significantly among CAP, COR and CAU sperms for each of the parameters studied (P < 0.01-P < 0.001). It is concluded that the differences observed among the three epididymal sperm populations are due to differences in the extent of susceptibility to decondensation in vitro and that this is dependent upon the variation in the -S-S-content of their chromatin during different stages of epididymal transit. All the parameters used (with the exception of fluorescence microscopy) can be quantified and as all of them show a similar dose dependency to DTT treatment, any one of these parameters can be conveniently used to determine the mature/immature status of the sperms voided. Application of such a method to determine the quality of sperms voided by man appears feasible.

Effect of dithiothreitol and sodium thioglycollate on protoplast production of saccharomyces cerevisae

Investigou-se a influência do ditiotreitol e do tioglicolato de sódio na produçäo de protoplastos de S. cerevisae. Resultados favoráveis foram obtidos a partir de 30 min. de digestäo, usando-se o ditioteitrol como pré-tratamento e adicionado à mesma soluçäo enzimática, inibiu a produçäo de protoplastos

Effect of anti-oxidants and adsorbents on tissue browning associated metabolism in Cocculus pendulus callus cultures.

Explants and callus of C. pendulus produced intense brown substances in the medium which caused necrosis. Various anti-oxidants (ascorbic acid, cysteine and dithiothreitol) and adsorbents (activated charcoal and polyvinyl pyrrolidone) were used in different concentrations to prevent browning of the tissues. These in MS medium affected differently the growth, colour and texture of the tissues. It was concluded that both peroxidase and phenolase were involved in the browning. Increased peroxidase activity and decreased phenolase activity were probably due to more peroxidative oxidation of phenols and unavailability of substrate for phenolase activity. This resulted in faster growth of tissues, which further reduced the phenolase activity.

Regulation of cadmium induced porphyria by ascorbic acid in chick embryos.

Sublethal doses of cadmium chloride (2.5, 5.0 and 10.0 mu mole/kg egg weight) were found to significantly alter the first two rate limiting enzymes of heme biosynthesis in chick embryos. Delta-aminolevulinic acid synthase activity was elevated by 2.05 and 2.11 fold with 5.0 and 10.0 mu moles of cadmium treatment respectively. However, this was reduced to 1.25 and 1.3 fold by the simultaneous administration of ascorbic acid. Blood delta-aminolevulinic acid dehydratase (ALA-D) activity was decreased by 48.4 and 55.0 per cent with 5.0 and 10.0 mu moles cadmium treatment respectively; in the presence of ascorbic acid only 18 and 24 per cent inhibition of ALA-D activity was observed. Further 1.39 and 2.08 fold accumulation of delta-aminolevulinic acid and 4.17 and 4.62 fold increase of blood porphyrins was observed in chick embryos treated with 5.0 and 10.0 mu moles cadmium respectively. This elevation of intermediate compounds of heme biosynthesis was effectively checked by the administration of ascorbic acid. Depletion of hepatic heme and free sulfhydryl level by cadmium were countered by the treatment of ascorbic acid. Hence, the present findings suggest the protective role of ascorbic acid against cadmium induced chemical porphyria in chick embryos.

Studies on the induction of cinnamic acid 4-hydroxylase in potato tuber.

The change in activity of cinnamic acid 4-hydroxylase (CA4H) in potato parenchyma tissue exposed to various conditions has been examined. Maximum induction of CA4H activity was obtained at 18 hr of incubation. Though CA4H induction can occur in dark, over 100% increase in enzyme activity was obtained on exposure of the tissue to light. Actinomycin D and cycloheximide inhibited the induction process. Mn2+, though known to cause an induction of CA4H in Jerusalem Artichoke, strongly inhibited potato CA4H induction. Dithiothreitol enhanced the CA4H activity due to either activation or protection of the enzyme. CA4H induction was significantly regulated at very low concentrations of trans-cinnamate and paracoumarate.