Antimony-induced oxidative stress and toxicity in cultured cardiac myocytes.

Cardiac myocytes were exposed to concentrations of potassium antimonyl tartrate (PAT) ranging from 1 to 1000 microM for 1 to 24 hr. Toxicity was assessed by measuring lactate dehydrogenase (LDH) release and by monitoring chronotropic depression. Lipid peroxidation was assessed by measuring the release of thiobarbituric acid reactive substances (TBARS). PAT produced a concentration- and time-dependent depression in chronotropy and an increase in the release of LDH and TBARS. A 4-hr exposure to 100 microM PAT stopped beating and induced significant increases in TBARS and LDH release in the myocyte cultures. The lipid peroxidation and LDH release induced by 100-200 microM PAT at 4 hr could be prevented by pretreatment of the cardiac myocytes with vitamin E or by the simultaneous addition of other antioxidants. Vitamin E continued to protect against lipid peroxidation up to 18 hr after the addition of 100 microM PAT, but failed to provide significant protection against LDH release at this time-point. Both 50 and 100 microM PAT decreased cardiac myocyte glutathione (GSH) levels after a 4-hr exposure. A series of thiol-containing compounds was evaluated for their effects on PAT toxicity. The addition of dithiothreitol, GSH, and 2-mercaptoethanol afforded some degree of protection against lipid peroxidation and LDH release up to 18 hr after the addition of 100 microM PAT. These results suggest that PAT induces lipid peroxidation in cultured cardiac myocytes but that other mechanisms may contribute to cell death with long-term exposures to PAT. Our results also suggest that PAT interacts with thiol-containing compounds.

Inhibition of intercellular communication in human keratinocytes by fractionated asphalt fume condensates.

Asphalt fume condensate (AFC) and chromatographically separated fractions have been shown to cause cancer in mouse skin. The levels of known carcinogenic initiators in these complex mixtures, however, are considered too low to account for their carcinogenic potency. It has been proposed that AFC may contain co-carcinogenic or tumor-promoting agents in addition to carcinogenic initiators. Modulation of gap junctional intercellular communication (GJIC) has been implicated as an important effect of tumor promoters. In this study, we examined the effect of five chromatographically generated fractions of AFC on GJIC in cultured human epidermal keratinocytes (HEK). HEK cells were exposed overnight to medium containing DMSO extracts of AFC fractions. GJIC was evaluated by dye-coupling of microinjected Lucifer Yellow CH. All AFC fractions produced a concentration-dependent inhibition of GJIC. The apparent potency of each fraction correlated with its relative polarity based on HPLC elution characteristics. Cells with reduced GJIC as a result of AFC fraction exposure were found to exclude propidium iodide, suggesting that inhibition of GJIC occurred in the absence of cell killing. However, significantly reduced culture DNA content was found following the overnight exposure to the highest concentrations of AFC fractions C, D and E.

Inhibition of rat heart mitochondrial electron transport in vitro: implications for the cardiotoxic action of allylamine or its primary metabolite, acrolein.

Allylamine (3-aminopropene) is a specific cardiac toxicant that causes aortic, valvular and myocardial lesions in many species. Myocardial necrosis can be observed 24 h after a single dose. Acute toxicity is believed to involve metabolism of allylamine to highly reactive acrolein (2-propenal). Allylamine has been shown to bind to mitochondria from aorta and heart, suggesting that the subcellular site of injury is at or near the mitochondrion. The present investigation compared the effect of allylamine and its primary metabolite, acrolein, on electron transport and oxidative phosphorylation in mitochondria isolated from rat heart (RHM). Both compounds weakly inhibited mitochondrial electron transport with either the combination of glutamate, malate, and malonate (GMM, NADH-linked) or succinate as substrate. Comparisons of the slopes of concentration-effect regression (range of concentrations tested, 0.20-2.0 mM) lines showed acrolein to have significantly greater inhibitory effects than allylamine (range of concentrations tested, 0.22-6.4 mM) on GMM oxidation, while no significant difference in the abilities of the compounds to inhibit succinate oxidation were observed, indicating site preferences for inhibitory action. The addition of an uncoupling agent could not reverse inhibition with either substrate system. These results indicate that both the parent compound and its proposed metabolite primarily inhibit electron transport with little direct effect on the coupling mechanism. The State III EC50 (effective concentrations for 50% inhibition of control mitochondrial enzyme activities) for allylamine (2.29 mM with succinate as substrate and 1.22 mM with GMM) and acrolein (0.80 mM with succinate as substrate and 0.39 mM with GMM) are probably too great to invoke the direct action of either the parent compound or its oxidized metabolite on mitochondrial electron transport as a primary mechanism in the cardiotoxic action of allylamine.

Transport of calcium by duodenum of spontaneously hypertensive rat.

The absorption of calcium by segments of duodenum obtained from spontaneously hypertensive (SH) rats and normotensive Wistar-Kyoto (WKy) rats was measured before and after the development of hypertension. The systolic blood pressure (SBP) of 5-wk-old SH rats (116 +/- 4 Torr) was significantly elevated above that of age-matched WKy rats (103 +/- 3 Torr) but was not at a level generally considered to be hypertensive. Values obtained for calcium transport [ratio of serosal-to-mucosal fluid 45Ca2+ concn (S/M ratio)] from everted duodenal sacs were similar between the two groups at this age. At 12 wk of age, SH rats exhibited a SBP (153 +/- 4 Torr) well above that of WKy controls (127 +/- 3 Torr), and calcium S/M ratios for duodenal sacs were significantly greater than the WKy control values. Similarly, the in vivo uptake of calcium in duodenal segments was significantly elevated in 12-wk-old SH rats compared with WKy controls. The administration of vitamin D3 or its metabolite, 25-hydroxycholecalciferol, had no detectable effect on duodenal transport of calcium in 12-wk-old SH or WKy rats. By comparison, 1,25-dihydroxycholecalciferol produced a significant increase in duodenal calcium transport both in vitro and in vivo in WKy but not in SH rats. The results indicate a distinct abnormality in the transport of calcium in the duodenum of SH rats, suggesting that the decrease in duodenal uptake of calcium that normally occurs with maturation is slow to develop in this rat strain.

The concentrations of ionic and total calcium in plasma of the spontaneously hypertensive rat.

The levels of ionic calcium in whole blood obtained from male spontaneously hypertensive rats (SHR) were slightly decreased compared with normotensive animals during the early stages of blood pressure elevation and in older (28-32 weeks) animals with established hypertension. During these intervals the total calcium concentrations of plasma were not significantly different although the ratios of ionic to total calcium were similar between SHR and control groups. In contrast with male and female SHR showed ionic calcium levels similar to normotensive control rats whereas total calcium concentrations in plasma increased with age and blood pressure elevation. The addition of calcium to plasma in 4- to 5-week-old SHR and normotensive rats revealed a buffering action in terms of the elevation in ionic calcium concentrations. The buffering capacity of plasma from normotensive animals did not change markedly but appeared to be greatly reduced in SHR by 17-25 weeks of age. Similarly, significant elevations in ionic calcium but not total calcium were observed following the injection of calcium in SHR compared with normotensive controls. The minor reductions in ionic calcium levels in blood seen in male SHR and the lowered ionic:total calcium ratios in female SHR may indicate enhanced binding or complexing of exogenous calcium. In view of these findings, the apparent reduction in in vitro and in vivo binding or complexing of exogenous calcium may reflect a decreased availability of sites for association with the calcium ion. The poor correlation between changes in blood pressure and ionic calcium suggested, however, that the disturbance in ionic or total calcium levels of plasma was not a primary causal factor in the development of hypertension.

Thermoregulatory ability of female rats during pregnancy and lactation.

Thermoregulatory ability of female rats was examined before pregnancy, during gestation, and during lactation. Thermoregulatory pattern, colonic temperature, evaporative water loss, and survival time were monitored during terminal heating (39.5 +/- 0.9 degrees C) designed to allow prolonged survival (3-4 h) with a sustained thermoregulatory effort. Results confirmed our previously reported observation of decreased thermoregulatory ability in lactating dams, with evidence suggesting thermoregulatory impairment during late gestation. Lactating dams displayed a type III thermoregulatory pattern, and established a rate of evaporative water loss effective for thermostasis at an elevated colonic temperature. However, survival time was significantly decreased compared to nonreproducing females. In contrast, prior heat acclimation tended to increase the survival time of lactating dams. It was concluded that the reduction in thermoregulatory ability observed in lactating dams was related to their inability to maintain a rate of evaporative water loss effective for thermostasis at an elevated colonic temperature.

Effect of heat exposure on in vitro intestinal transport and utilization of glucose in the rat.

Intestinal transport and utilization of glucose were studied in vitro in chronically heat-exposed (Ta = 34 degrees C) rats. Despite mucosal tissue per centimetre being significantly reduced following 7 and 14 days of exposure, no differences from control values were noted in full serosal to mucosal (S/M) fluid glucose concentrations ratios obtained from everted sac preparations. Measurements on 40-cm jejunal segments perfused in vitro at days 13-15 showed a reduction in the rate of glucose absortpion per centimetre of intestine, while glucose absorption per gram of tissue was unaltered in heat-exposed rats. At the same time, glucose secretion at the serosal surface was unchanged as calculated per centimetre of intestine and increased when calculated per gram of dry tissue. The utilization of glucose by the intestine was found to be significantly reduced whether expressed for intestinal length or for intestinal weight. In addition, the glucose concentration of the tissue water was elevated in intestinal segments of heat-exposed rats after 60 min of perfusion. The decrease in glucose uptake per centimetre of intestine was attributed to the decrease in mucosal tissue. Elevated tissue glucose concentration and enhanced translocation of glucose to the serosal surface were attributed, at least in part, to the decreased rate of glucose metabolism observed in intestines of heat-exposed rats.

Periodic, short-term heat exposure and reproductive function in male and female rats.

Reproductive function of male and female rats was examined in relation to periodic, short-term heat treatment. Daily exposure to an environmental temperature of 38.2 degrees C for 55 min elevated rectal temperatures to 39.9 and 41.2 degrees C in male and female rats, respectively. Heat exposure tended to decrease copulation in males cohabitated with unheated females. The rate of conception was affected similarly, and fetal survival tended to be reduced by paternal heat treatment. Estrous cycles were disrupted initially in heat-exposed females, but the rate of copulation and conception of females cohabitated with unheated males was unaltered by heat treatment. However, maternal heat exposure impaired prenatal survival and growth. During lactation, a high incidence of maternal and pup deaths was observed at approximately 14 days postpartum. Maternal deaths were coincident with a decrease in thermoregulatory ability and rectal temperatures exceeding 42 degrees C.