A pressing need exists to develop and validate molecular biomarkers to assess the early effects of chemical agents, both individually and in mixtures. This is particularly true for new and chemically intensive industries such as the semiconductor industry. Previous studies from this laboratory and others have demonstrated element-specific alterations of the heme biosynthetic pathway for the III–V semiconductors gallium arsenide (GaAs) and indium arsenide (InAs) with attendant increased urinary excretion of specific heme precursors. These data represent an example of a metabolomic biomarker to assess chemical effects early, before clinical disease develops. Previous studies have demonstrated that the intratracheal or subcutaneous administration of GaAs and InAs particles to hamsters produces the induction of the major stress protein gene families in renal proximal tubule cells. This was monitored by 35-S methionine labeling of gene products followed by two-dimensional gel electrophoresis after exposure to InAs particles. The present studies examined whether these effects were associated with the development of compound-specific proteinuria after 10 or 30 days following subcutaneous injection of GaAs or InAs particles in hamsters. The results of these studies demonstrated the development of GaAs- and InAs-specific alterations in renal tubule cell protein expression patterns that varied at 10 and 30 days. At the 30-day point, cells in hamsters that received InAs particles showed marked attenuation of protein expression, suggesting inhibition of the stress protein response. These changes were associated with GaAs and InAs proteinuria patterns as monitored by two-dimensional gel electrophoresis and silver staining. The intensity of the protein excretion patterns increased between the 10- and 30-day points and was most pronounced for animals in the 30-day InAs treatment group. No overt morphologic signs of cell death were seen in renal tubule cells of these animals. Western blot analyses of the urines with antibodies to the 32-, 70-, and 90-kDa stress protein families did not show the presence of these molecules, indicating that these proteins were not excreted in the urine samples. These data suggest that the observed proteinuria patterns were not a result of cell death and that the observed chemical-specific proteinurias were produced before marked cellular toxicity. These findings suggest a hypothesis involving GaAs and InAs interference with stress protein chaperoning of reabsorbed proteins for proteosomic degradation and the probable chaperoning of damaged intracellular proteins from renal proximal tubule cells into the urinary filtrate. Overall, the results of these studies provide further information on the nephrotoxicity of these semiconductor compounds. They also suggest the use of two-dimensional gel electrophoresis with silver staining of urinary protein patterns as a potentially useful proteomic approach to renal damage early in relation to intracellular proteotoxicity in kidney tubule cells.

The effects of indium and arsenic on the heme biosynthetic pathway have been well documented but the effects of indium arsenide (InAs), the next possible generation of the III-V semiconductors, are unknown. Male Syrian golden hamsters were given s.c. injections of sodium arsenite (As3+), indium chloride (In3+) or indium arsenide (InAs). Erythrocyte δ-aminolevulinic acid dehydratase (ALAD) activity was inhibited in all exposure groups, while hepatic ALAD activity was not significantly changed. In contrast, the activity of renal ALAD was found to be statistically decreased by As3+ at 10 days, but increased at 30 days, while In3+ and InAs inhibited this enzyme activity at all time points. In vitro studies showed that hepatic ALAD activity was more sensitive to In3+ than As3+, suggesting that the effects of InAs in vivo on this enzyme are due primarily to the In rather than the As moiety. Studies of urinary porphyrin excretion patterns in animals treated with InAs showed marked, early 2–4-fold increases in the excretion of the penta-, hexa- and heptacarboxyl porphyrin at 1–5 days which continued through day 30 of the study. In contrast, there was a slow and steady rise in the excretion of coproporphyrin I and III which reached a maximum at day 30. The results of these studies indicate that both the In and As moieties of InAs are biologically active following InAs exposure and that the enzymes in the heme pathway, such as ALAD, may have great utility as markers of exposure/toxicity for these agents.

Selective inhibition of mammalian δ-aminolevulinic acid dehydratase (ALAD; porphobilinogen synthetase, EC 4.2.1.24) by Pb serves as a important biological marker of chemical exposure and injury. Water-borne exposure of Pb has been shown to inhibit ALAD in the blood and liver of several fish species. This study was undertaken to evaluate the relative susceptibility of fish hepatic ALAD to Pb inhibition in vitro and to characterize this enzyme as determined by a combination of biochemical and immunological techniques. The reaction rate and assay sensitivity were found to increase as a function of temperature and duration of incubation. The results of these studies demonstrated that the IC50 (17.3 μM) for Pb inhibition of fish liver ALAD activity was 40-times higher than those values reported for rats (0.31–0.4 μM Pb). Kinetic analyses of fish hepatic ALAD activities indicated a Km of 0.043 ± 0.005 mM ALA and a Vmax of 2.57 ± 0.218 nmol porphobilinogen (PBG)/h/mg of protein. Further in vitro studies showed no activation of fish hepatic ALAD by Zn and only moderate inhibition by EDTA relative to rat ALAD. Western blot analyses using rabbit polyclonal antibodies directed to purified human erythrocyte ALAD suggested faint immunological cross-reactivity. These studies indicate that fish hepatic ALAD is biochemically distinct from the mammalian enzyme and that Zn is apparently not a cofactor for this enzyme and/or that the binding of any metal cofactor to the enzyme is extremely stable.

The present study reports the isolation and partial characterization of a lead-binding protein (PbBP) in the liver of the channel catfish (Ictalurus punctatus). The protein has a molecular weight of 10 kDa as determined by SDS polyacrylamide gel electrophoresis and contains relatively large amounts of glycine (18.3%), aspartic acid (10.2%) and serine (15.1%). Western blot studies conducted using polyclonal antibodies to the rat renal PbBP and metallothionein (MT) showed no cross-reactivity, suggesting that the fish hepatic protein is immunologically distinct from these low-molecular weight metal-binding proteins in mammals.

These studies were conducted to assess alterations in renal tubule cell gene expression following in vivo exposure to the semiconductor elements indium (In), arsenic (As) or indium arsenide (InAs). Alterations in proximal tubule cell gene expression were monitored at similar tissue concentrations of In or As at various time-points following single subcutaneous (sc) injections of In, As or InAs at 0, 10 and 30 days (In: 1.5 mg/kg; As, 3 mg/kg or 0.3 mg/k; and InAs: 1000 mg/kg). Protein synthesis as monitored by incorporation of 35S methionine was not statistically increased over the 30-day period following sc injection of As, In or InAs relative to controls. Two dimensional--SDS/polyacrylamide gel electrophoresis showed that exposure to InAs stimulated the synthesis of a number of proteins with molecular masses of < 10, 18, 28, 32, 38, 42, 58, 70, 98 KDa. Exposure to As produced an increase in the expression of thirteen gene products. Indium produced similar changes at the 10-day time-point, but increased tissue accumulation of this element at 30 days markedly suppressed the stress protein response. These data indicate that induction of these specific gene expression patterns may be useful as early indicators for assessing exposure to InAs, or inorganic As, while suppression of these responses by In suggests a compromise in this basic protective mechanism.

Lead-binding proteins have previously been isolated from rat and human target tissues. These molecules have shown to possess molecular masses in the general range of 10,000-30,000 daltons. The proteins are acidic in nature and rich in aspartic and glutamic amino acid residues. The molecules in rodents appear to play several important roles in mediating the low dose toxicity of lead in the kidney and brain. Preliminary studies presented in this report indicate that monkeys also possess similar proteins in the kidney and brain, thus providing a biochemical "bridge" in a non-human primate between rodent models and humans. Further, the excretion of these molecules into the urine of rodents increases with lead exposure, suggesting that may also prove useful as biomarkers of lead exposure in humans and monkeys once the dose-range and mechanism(s) of this phenomenon are further defined. Such studies should provide valuable risk assessment information for determining why individuals vary in their susceptibility to lead toxicity.

Of the semiconductor metals, only arsenic has been extensively studied as a human carcinogen and systemic toxicant. Recent studies have shown, however, that gallium, arsenic, and indium are capable of producing marked alterations in cellular gene products. After acute in vivo administration indium and thallium have been shown to produce decreases in the activity of some drug-metabolizing enzymes dependent on cytochrome P-450; therefore these metals would be capable of interfering with the metabolism of organic carcinogens. Selenium is essential for the activity of the enzyme glutathione peroxidase, which modulates the active intermediates generated by drug-metabolizing enzyme systems. Germanium produces toxicity in a number of organ systems. Antimony produces lung and circulatory system effects. Overall, available data suggest that these metals or metalloids are capable of biologically altering several cellular defense mechanisms involved in the carcinogenic process and that further studies are needed to determine the associated risks.