James S. Woods

Altered regulation of mammalian hepatic heme biosynthesis and urinary porphyrin excretion during prolonged exposure to sodium arsenate

Continuous prolonged exposure to sodium arsenate at 20, 40, or 85 ppm in the drinking water resulted in depression of hepatic δ-aminolevulinic acid (ALA) synthetase and heme synthetase, the first and last enzymes in heme biosynthesis, respectively, in both rats and mice. ALA synthetase was maximally depressed to approximately 80% of control values at 40 ppm in both species, whereas heme synthetase activity was maximally decreased to 63 and 75% of control at 85 ppm in rats and mice, respectively. Uroporphyrinogen I synthetase, the third enzyme in heme biosynthesis, was increased at all doses in the mouse, whereas ALA dehydratase, the second heme biosynthetic pathway enzyme, was unaltered in either species. Concomitantly, urinary uroporphyrin concentrations were elevated by as much as 12 times, and coproporphyrin by as much as nine times, the control values in the rat. Similar patterns of elevated porphyrin excretion were seen in the mouse. In contrast, no changes were observed in the activities of cytochrome oxidase or cytochrome P-450, indicators of mitochondrial and microsomal hemoprotein function, respectively. These results demonstrate that prolonged exposure to low levels of arsenic results in selective alteration of hepatic heme biosynthetic pathway enzymes, with concomitant increases in urinary porphyrin concentrations. These changes, which are uniquely characterized by a predominant increase in uroporphyrin over coproporphyrin concentrations, occur independently of changes in hepatic hemoprotein function and may thus serve as a specific indicator of pretoxic arsenic exposure.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) effects on hepatic microsomal cytochrome P-448-mediated enzyme activities☆☆☆

Abstract The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatic microsomal mixed function oxidase (MFO) enzyme systems were examined in female rats. Although TCDD had little effect on NADPH-cytochrome c reductase activity and cytochrome P-450 content, the activities of the cytochrome P-448-mediated enzymes benzo[α]pyrene hydroxylase, ethoxyresorufin O-deethylase, and biphenyl 2-hydroxylase were greatly increased. Three months after a single oral dose of 2 μg/kg TCDD, the cytochrome P-450 content and benzo[α]pyrene hydroxylase and ethoxyresorufin O-deethylase activities were still significantly increased. In addition, the microsomal metabolism of the novel substrate 4,4′-dimethylbiphenyl was greatly increased by TCDD pretreatment. Low dose studies revealed that the ED50 of TCDD induction of benzo[α]pyrene hydroxylase was 0.63 μg/kg and the lowest dose of TCDD which caused a significant increase in enzyme activity was 0.002 μg/kg. Studies in which [1,6-3H]TCDD was used to determine the extent of hepatic uptake of orally administered TCDD at the lowest effective dose of 0.002 μg/kg lead to the estimate that only 65 molecules of TCDD per hepatocyte were required to produce a measurable increase in benzo[α]pyrene hydroxylation. These results attest to the specificity and persistence of TCDD in the induction of cytochrome P-448-mediated enzyme activities in rat liver. The small number of molecules required to induce benzo[α]pyrene hydroxylase suggests that TCDD is among the most potent MFO-inducing agents yet demonstrated in mammalian liver.

Ultrastructural and biochemical changes in renal mitochondria during chronic oral methyl mercury exposure: The relationship to renal function

Abstract The report describes the effects of chronic oral methyl administration on renal tubule cell mitochondria in relation to standard tests of renal function and porphyrinuria associated with prolonged methyl mercury exposure. Male rats were given access to drinking water containing 0, 3, 5, or 10 ppm mercury as methyl mercuric hydroxide (MMH) for 6 weeks. In situ swelling of renal proximal tubule cell mitochondria observed by electron microscopy was associated with decreases in respiratory control ratios of cortical mitochondria. The specific activity of monoamine oxidase, which is localized on the outer mitochondrial membrane, showed a dose-related decrease in MMH-treated animals. Cytochrome oxidase, which was used as a marker enzyme for the inner mitochondrial membrane, showed a more moderate decrease in specific activity. In contrast, δ-aminolevulinic acid synthetase, which is loosely bound to the inner mitochondrial membrane and is the rate-limiting enzyme in the heme biosynthetic pathway, showed a 2- to 2.5-fold increase in specific activity. Malate dehydrogenase, which was used as a marker enzyme for the mitochondrial matrix, showed no change in activity. The above findings were associated with elevated concentrations of heme precursors in the urine of MMH-treated animals, but no changes in blood urea nitrogen, serum creatinine values, or increased urinary excretion of lysozyme. These results are discussed in regard to the specificity of the effects of MMH on renal mitochondrial membranes and the potential utility of these effects in the assessment of biological responses to mercury exposure prior to the onset of overt clinical toxicity.

The effects of prolonged oral arsenate exposure on liver mitochondria of mice: Morphometric and biochemical studies☆

Exposure of mice to arsenic in drinking water at concentrations of 0, 20, 40, or 85 ppm for 6 weeks resulted in mild swelling of hepatocyte mitochondria, binucleate cells, and a slight but not statistically significant increase in the overall relative volume-density of the nuclear compartment, as determined by ultrastructural morphometry. Despite only slight changes in morphometric parameters, hepatocyte mitochondria in arsenate-treated mice showed a significant decrease in pyruvate/malate, but not succinate-mediated state 3 respiration and respiratory control ratios, and decreases in PO ratios with both substrate types. Mitochondria from arsenate treated mice also showed a significant dose-related increase of up to 175% of control in the specific activity of monoamine oxidase, which was used as an outer membranal marker enzyme. Cytochrome oxidase and Mg2+-ATPase, which are localized on the inner mitochondrial membrane showed statistically nonsignificant increases in specific activity of up to 150 and 120% of control, respectively. In contrast, malate dehydrogenase, which was used as a marker for enzymatic activity in the mitochondrial matrix, showed no significant alteration in activity. Ultrastructural and biochemical data were related to liver concentrations of total arsenic. The above studies demonstrate that, despite little quantitative physical alteration of the mitochondrial compartment, significant changes in various membrane-associated biochemical functions may occur in mouse liver mitochondria during prolonged arsenate exposure.

Altered regulation of hepatic heme metabolism by indium chloride

Abstract These studies were conducted to investigate the effects of indium, a hepatotoxic group III A metal, on cellular heme metabolism in rat liver. Treatment of rats with aqueous solutions of InCl 3 produced rapid and pronounced changes in hepatic heme biosynthetic and degradative functions, characterized by depression of synthesis of ALA synthetase and induction of heme oxygenase, the rate-limiting enzymes in these processes, respectively. These changes were accompanied by a marked reduction in microsomal heme concentrations and a decrease in microsomal, but not mitochondrial, heme-dependent oxidative functions in the liver. The relationship of these effects to the regulation of heme metabolism by metals of known physiological importance remains to be determined. However, these observations suggest the presence of a regulatory process for heme metabolism which is susceptible to alteration by a variety of metallic substances including those of no known physiological significance. The rapidity and magnitude of the changes in heme metabolic parameters observed in these studies suggests that these effects may represent an initial response to indium-induced toxicity in liver cells.

Selective inhibition of δ-aminolevulinic acid dehydratase by indium chloride in rat kidney: Biochemical and ultrastructural studies

Abstract Indium chloride, a nephrotoxic and porphyrogenic chemical, exerts a tissue-specific inhibition of δ-aminolevulinic acid (ALA) dehydratase in rat kidney. This effect appears to be related to the preferential accumulation of indium by the kidney proximal tubule cell and to the selective localization of indium within the cytosolic fraction of that cell type, as assessed by electron microscopy. The reversal of indium-induced inhibition of renal ALA dehydratase both in vivo and in vitro by zinc suggests that indium acts by binding with sulfhydryl groups of the enzyme. The synergistic effects of lead and indium with respect to inhibition of renal ALA dehydratase suggest that exposure to weak sulfhydryl inhibitors such as the group III metals may enhance the renal toxicity of other metals which may not in themselves exert overt toxic effects in that organ.

Studies on the action of porphyrinogenic trace metals on the activity of hepatic uroporphyrinogen decarboxylase

Abstract Trace metals which produce experimental uroporphyrinuria in animals during prolonged exposure inhibit uroporphyrinogen (uro) decarboxylase in rat liver extracts in , vitro . Inhibitory effects are prevented by sulfhydryl reagents, suggesting metal binding to sulfhydryl groups of the enzyme as the likely mode of action. Mercury, the most potent of the metals tested with respect to sulfur binding kinetics, produces the greatest inhibition of enzyme activity. In contrast, iron, which is considered to play a role in the etiology of porphyria cutanea tarda (PCT) via inhibition of uro decarboxylase, did not inhibit the enzyme in the present test system, suggesting an indirect mode of action in , vivo . These results suggest that direct inhibition of uro decarboxylase underlies uroporphyrinuria produced by prolonged trace metal exposure. Experimental inhibition of uro decarboxylase by trace metals may serve as a model for studying metal-induced uroporphyrinuria and PCT in humans.

Alteration of hepatocellular structure and function by thallium chloride ultrastructural morphometric and biochemical studies

The effects of thallium chloride (TlCl3.4H2O) on hepatocyte structure and function were studied in male rats at 16 hr following treatment by ip injection with doses of 0, 50, 100, and 200 mg/kg. Ultrastructural examination of hepatocytes from thallium-treated rats showed a dose-related loss of ribosomes from the endoplasmic reticulum and proliferation of the rough endoplasmic reticulum segment. Generalized mitochondrial swelling and increased numbers of electron-dense autophagic lysosomes were also observed. Morphometric analysis of hepatocytes from thallium-treated rats disclosed a 3-fold increase in the volume density of the lysosomal compartment and a 1.3-fold increase in the volume density of mitochondrial. Surface density measurements of mitochondrial and endoplasmic reticulum membranes showed dose-related increases in the surface density of both inner and outer mitochondrial membranes as well as of the rough endoplasmic reticulum. These structural changes were associated with pronounced increases in the specific activities of the mitochondrial membrane-associated enzymes monoamine oxidase and ferrochelatase to 145 and 144% of control values, respectively, and a 42% decrease in the activity of aminolevulinic acid (ALA) synthetase. Similarly, structural alteration of the endoplasmic reticulum in thallium-treated rats was associated with concomitant impairment of the microsomal enzymes NADPH cytochrome c (P-450) reductase, aniline hydroxylase, and aminopyrene demethylase to a maximum of 49, 43, and 77% of activities seen in untreated controls, respectively. In contrast, the non-membrane-bound enzymes malate dehydrogenase, ALA dehydratase, and uroporphyrinogen I synthetase were unaltered in vivo following thallium treatment at any doses. These results indicate that thallium-induced alteration of hepatic biochemical processes may arise from physical disruption of the membranal integrity of subcellular organelles with which those processes are functionally associated. These findings are consistent with those from previous studies in demonstrating a positive quantitative correlation between metal-induced subcellular organelle membrane structural injury and impairment of associated biological functions in vivo.

Developmental aspects of hepatic heme biosynthetic capability and hematotoxicity

Abstract δ-Aminolevulinic acid (ALA) synthetase is considered to be rate-limiting in heme biosynthesis in normal adult mammalian liver. However, under certain pharmacological or pathological circumstances, other enyzmes of the heme biosynthetic pathway have been shown to be limiting in this process. In the current studies, the developmental patterns of ALA dehydratase, uroporphyrinogen I synthetase and heme synthetase were measured in rat liver, and the potential influence of these enzymes on heme biosynthetic capability under various conditions in adult, neonatal and fetal liver was assessed. In addition, a comparison of the activities of these enzymes with those of ALA synthetase was made as a means of assessing the relative influence of hematotoxic agents on hepatic heme biosynthetic capability at different stages of development.

Studies on the mechanisms of thalliummediated inhibition of hepatic mixed function oxidase activity: Correlation with inhibition of NADPH-cytochrome c (P-450) reductase

Thallium (TlCl3) administration to rats produced a dose-dependent loss of hepatic NADPH-cytochrome c (P-450) reductase and microsomal mixed function oxidase activities within 2-4 hr following treatment. These changes occurred independently of apparent effects on microsomal heme or cytochrome P-450 content, both of which remained unchanged with respect to control levels despite transient inhibition of delta-aminolevulinic acid (ALA) synthetase and induction of heme oxygenase. These results are consistent with the recognized properties of thallium as both a flavoprotein antagonist and sulfhydryl inhibitor and differ uniquely from the action of other metals which impair mixed function oxidase activity through compromise of heme biosynthesis and heme depletion. The potential utility of thallium compounds in further evaluating the functional characteristics of NADPH-cytochrome c (P-450) reductase and its role in microsomal oxidative processes is suggested from these observations.