Insights into possibilities for grouping and read-across for nanomaterials in EU chemicals legislation.

This paper presents a comprehensive review of European Union (EU) legislation addressing the safety of chemical substances, and possibilities within each piece of legislation for applying grouping and read-across approaches for the assessment of nanomaterials (NMs). Hence, this review considers both the overarching regulation of chemical substances under REACH (Regulation (EC) No 1907/2006 on registration, evaluation, authorization, and restriction of chemicals) and CLP (Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures) and the sector-specific pieces of legislation for cosmetic, plant protection and biocidal products, and legislation addressing food, novel food, and food contact materials. The relevant supporting documents (e.g. guidance documents) regarding each piece of legislation were identified and reviewed, considering the relevant technical and scientific literature. Prospective regulatory needs for implementing grouping in the assessment of NMs were identified, and the question whether each particular piece of legislation permits the use of grouping and read-across to address information gaps was answered.

White paper on the promotion of an integrated risk assessment concept in European regulatory frameworks for chemicals.

The vision of a sustainable and safe use of chemicals to protect human health, preserve the environment and maintain the ecosystem requires innovative and more holistic approaches to risk assessment (RA) in order to better inform decision making. Integrated risk assessment (IRA) has been proposed as a solution to current scientific, societal and policy needs. It is defined as the mutual exploitation of environmental risk assessment (ERA) for human health risk assessment (HHRA) and vice versa in order to coherently and more efficiently characterize an overall risk to humans and the environment for better informing the risk analysis process. Extrapolating between species which are relevant for HHRA and ERA requires a detailed understanding of pathways of toxicity/modes of action (MoA) for the various toxicological endpoints. Significant scientific advances, changes in chemical legislation, and increasing environmental consciousness have created a favourable scientific and regulatory environment to develop and promote the concept and vision of IRA. An initial proof of concept is needed to foster the incorporation of IRA approaches into different chemical sectorial regulations and demonstrate their reliability for regulatory purposes. More familiarity and confidence with IRA will ultimately contribute to an overall reduction in in vivo toxicity testing requirements. However, significant progress will only be made if long-term support for MoA-related research is secured. In the short term, further exchange and harmonization of RA terminology, models and methodologies across chemical categories and regulatory agencies will support these efforts. Since societal values, public perceptions and cultural factors are of increasing importance for the acceptance of risk analysis and successful implementation of risk mitigation measures, the integration of socio-economic analysis and socio-behavioural considerations into the risk analysis process may help to produce a more effective risk evaluation and consideration of the risks and benefits associated with the use of chemicals.

Perspectives for integrating human and environmental risk assessment and synergies with socio-economic analysis.

For more than a decade, the integration of human and environmental risk assessment (RA) has become an attractive vision. At the same time, existing European regulations of chemical substances such as REACH (EC Regulation No. 1907/2006), the Plant Protection Products Regulation (EC regulation 1107/2009) and Biocide Regulation (EC Regulation 528/2012) continue to ask for sector-specific RAs, each of which have their individual information requirements regarding exposure and hazard data, and also use different methodologies for the ultimate risk quantification. In response to this difference between the vision for integration and the current scientific and regulatory practice, the present paper outlines five medium-term opportunities for integrating human and environmental RA, followed by detailed discussions of the associated major components and their state of the art. Current hazard assessment approaches are analyzed in terms of data availability and quality, and covering non-test tools, the integrated testing strategy (ITS) approach, the adverse outcome pathway (AOP) concept, methods for assessing uncertainty, and the issue of explicitly treating mixture toxicity. With respect to exposure, opportunities for integrating exposure assessment are discussed, taking into account the uncertainty, standardization and validation of exposure modeling as well as the availability of exposure data. A further focus is on ways to complement RA by a socio-economic assessment (SEA) in order to better inform about risk management options. In this way, the present analysis, developed as part of the EU FP7 project HEROIC, may contribute to paving the way for integrating, where useful and possible, human and environmental RA in a manner suitable for its coupling with SEA.

Tuberin phosphorylation regulates its interaction with hamartin. Two proteins involved in tuberous sclerosis.

Hamartin and tuberin are products of the tumor suppressor genes, TSC1 and TSC2, respectively. When mutated, a characteristic spectrum of tumor-like growths develop resulting in the syndrome of tuberous sclerosis complex. The phenotypes associated with TSC1 and TSC2 mutations are largely indistinguishable suggesting a common biochemical pathway. Indeed, hamartin and tuberin have been shown to interact stably in vitro and in vivo. Factors that regulate their interaction are likely critical to the understanding of disease pathogenesis. In this study, we showed that tuberin is phosphorylated at serine and tyrosine residues in response to serum and other factors, and it undergoes serial phosphorylation that can be detected by differences in electrophoretic mobilities. A disease-related TSC2 mutation (Y1571H) nearly abolished tuberin phosphorylation when stimulated with pervanadate. Expression of this mutant tuberin caused a marked reduction in TSC1-TSC2 interaction compared with wild-type protein and significantly curtailed the growth inhibitory effects of tuberin when overexpressed in COS1 cells, consistent with a loss of function mutation. Examination of a second pathologic mutation, P1675L, revealed a similar relationship between limited phosphorylation and reduced interaction with hamartin. Our data show for the first time that 1) tuberin is phosphorylated at tyrosine and serine residues, 2) TSC1-TSC2 interaction is regulated by tuberin phosphorylation, and 3) defective phosphorylation of tuberin is associated with loss of its tumor suppressor activity. These findings suggest that phosphorylation may be a key regulatory mechanism controlling TSC1-TSC2 function.

Expression of the casein kinase 2 subunits in Chinese hamster ovary and 3T3 L1 cells provides information on the role of the enzyme in cell proliferation and the cell cycle.

In order to investigate the in vivo functions of protein kinase CK2 (CK2), the expression of Myc-tagged versions of the subunits, Myc-CK2alpha and Myc-CK2beta, was carried out in Chinese hamster ovary cells (CHO cells) and in 3T3 L1 fibroblasts. Cell proliferation in these cells was examined. CHO cells that transiently overexpressed the Myc-CK2beta subunit exhibited a severe growth defect, as shown by a much lower value of [(3)H]thymidine incorporation than the vector controls, and a rounded shrunken morphology. In contrast, cells overexpressing Myc-tagged CK2alpha showed a slightly but consistently higher value of [(3)H]thymidine incorporation than the controls. The defect in cell growth and changes in morphology caused by Myc-CK2beta overexpression were partially rescued by coexpression of Myc-tagged CK2alpha. In parallel to the studies in CHO cells, the stable transfection of Myc-CK2alpha and Myc-CK2beta subunits was achieved in 3T3 L1 fibroblast cells. Similarly, the ectopic expression of Myc-CK2beta, but not Myc-CK2alpha, caused a growth defect. By measuring [(3)H]thymidine incorporation, it was found that expression of Myc-CK2beta prolonged the G(1) phase and inhibited up-regulation of cyclin D1 expression during G(1). In addition, a lower mitotic index and lower mitotic cyclin-dependent kinase activities were detected in Myc-CK2beta-expressing cells. Detailed analysis of stable cells that were synchronously released into the cell cycle revealed that the expression of Myc-CK2beta inhibited cells entering into mitosis and prevented the activation of mitotic cyclin-dependent kinases. Taken together, results from both transient and stable expression of CK2 subunits strongly suggest that CK2 may be involved in the control of cell growth and progression of the cell cycle.

Psychiatrists meet on the web: the chance for a virtual congress. Results and experiences from the First International Internet Congress on Psychiatry 1997.

From December 1-31, 1997, the Department of Psychiatry and Psychotherapy in co-operation with the Department of Information Technology, University of Tübingen, Germany, organised the first virtual congress on psychiatry in the Internet. The congress was aimed at facilitating exchange of results of psychiatric studies and ideas and at stimulating discussion among interested colleagues. Almost 100 participants from 17 countries on four continents took part in this event. Sixteen contributions were presented and discussed. The problems and opportunities of this medium in the organisation and running of congresses are presented and discussed. The experience gained in this congress suggests that the Internet will find increasing use as a medium for medical congresses within the next few years.

Changes in the liver protein pattern of female Wistar rats treated with the hypoglycemic agent SDZ PGU 693.

SDZ PGU 693 acts as a hypoglycemic agent by stimulating glucose utilisation in insulin-sensitive peripheral tissues, such as skeletal muscle and fat. In a 28 day toxicity study the compound was found to induce hepatocellular hypertrophy in Wistar rats treated with 300 mg/kg/day. To gain insights into the pathomechanism of these alterations, aliquots of liver samples from control and treated female Wistar rats were separated by two-dimensional protein gel electrophoresis and the digitized images of the protein patterns were searched for protein abundance changes. Significant treatment-related quantitative changes (P < 0.001) were found in 29 liver proteins. Major increases were observed in several microsomal proteins, including NADPH cytochrome P-450 reductase, cytochrome b5 and serine protease inhibitor. The changes in the cytochrome related enzymes, both known co-factors of the P-450 enzyme system, strongly suggest that SDZ PGU 693 induces microsomal proliferation and induction of the P-450 enzyme system. Decreases were observed in a series of mitochondrial proteins, such as F1ATPase-delta subunit and ornithine aminotransferase precursor as well as in several cytosolic proteins such as the liver fatty acid binding protein, arylsulfotransferase and the senescence marker protein-30. The changes in F1ATPase-delta subunit and liver fatty acid binding protein together suggest a down-regulation of the mitochondrial liver fatty acid metabolism, likely reflecting the pharmacological action of the compound. These results show that SDZ PGU 693 produces a complex pattern of gene expression changes which give insights into the molecular mechanisms of both its pharmacological action and a toxic response.

New insights into cyclosporine A nephrotoxicity by proteome analysis.

Using two-dimensional gel electrophoresis (2-DE), we recently discovered an association between decreased calcium-binding protein, calbindin-D 28 kDa, urinary calcium wasting and intratubular corticomedullary calcifications in rat kidney. This observation prompted us to investigate kidney tissues of other species, including man. In this paper we show that in dogs and monkeys, which are generally devoid of cyclosporine A (CsA)-mediated nephrotoxicity, renal calbindin levels were not affected by the CsA treatment whereas in CsA-treated human kidney-transplant recipients with renal vascular or tubular toxicity, a marked decrease in renal calbindin-D 28 kDa protein level was found in most of the kidney biopsy sections. The present results strongly suggest that calbindin is a marker for CsA-nephrotoxicity. The discovery of calbindin-D 28 kDa being involved in CsA toxicity has evolved from the application of 2-DE and has not been reported previously, proving that proteomics can provide essential information in mechanistic toxicology. Considering the current improvements in proteome methods it is expected that high throughput proteomics will become an indispensable tool in preclinical safety testing.

Cyclosporine A-induced decrease in calbindin-D 28 kDa in rat kidney but not in cerebral cortex and cerebellum.

Recently, we reported that in rat, cyclosporine A (CsA) markedly decreases the levels of calbindin-D (CABP-D) 28 kDa in kidney. CABP-D 28 kDa is a calcium-binding protein which is highly expressed in calcium-transporting tissues such as kidney or brain. In this study, we investigated whether, in addition to the kidney, CsA also has an effect on CABP-D 28 kDa in rat brain. Three groups of male Wistar rats received 15 mg/kg/day or 50 mg/kg/day of CsA orally for 12 days, whereas controls received vehicle solution for the same period. CABP-D 28-kDa protein and CsA were quantified in homogenates of kidney, cerebral cortex and cerebellum, and the localization of CABP-D 28 kDa was assessed in the different tissue sections by immunohistochemistry. In kidney, CABP-D 28 kDa was strongly and dose dependently decreased, and was located in tubular epithelial cells. In brain, CABP-D 28 kDa was not changed and was mainly located in pyramidal cells of the cortex and in cerebellum exclusively in Purkinje cells. High CsA concentrations were measured in kidney, more than 17-fold greater than those found in cortex. In cerebellum, CsA was below the limit of detection. These data suggest that at clinically relevant doses, CsA may not affect CABP-D 28-kDa levels in brain.

Hepatocellular effects of cyclosporine A and its derivative SDZ IMM 125 in vitro.

The novel immunosuppressive drug O-hydroxyethyl-D(Ser)8-cyclosporine (SDZ IMM 125) and cyclosporine A (CyA) were compared in different in vitro models with respect to hepatocellular side effects. SDZ IMM 125 was less lipophilic than CyA and also decreased liposomal membrane anisotropy less. Furthermore, SDZ IMM 125 increased Na+ and Ca++ permeability across the liposomal membranes significantly more than CyA. The uptake of CyA and SDZ IMM 125 into freshly isolated rat hepatocytes was neither saturable, Na+ dependent or temperature sensitive, nor could it be inhibited vice versa, indicating passive diffusion. The diffusion coefficient of CyA was about two times higher than that of SDZ IMM 125, reflecting its higher lipophilicity. In primary hepatocyte monolayers the cellular concentrations of CyA were about two times higher than that of SDZ IMM 125. As an indicator of cholestasis the saturable uptake of cholyltaurine into isolated cells was found to be apparently competitively inhibited to the same extent by both compounds. In isolated perfused rat livers SDZ IMM 125 caused a significantly greater decrease in bile flow than did CyA. Release of lactate dehydrogenase from hepatocyte primary cultures and from isolated perfused livers were determined as parameter of cell damage. In both systems the cytotoxicity of SDZ IMM 125 was significantly higher than that of CyA. The data suggest that SDZ IMM 125 causes greater cholestatic and cytotoxic effects than CyA at equimolar cellular exposure.

Decrease in kidney calbindin-D 28kDa as a possible mechanism mediating cyclosporine A- and FK-506-induced calciuria and tubular mineralization.

The use of the immunosuppressant cyclosporine A (CsA) is limited by its adverse renal effects. Most recently, we reported that the drug markedly decreases the levels of the calcium-binding protein calbindin-D 28kDa in kidneys of male Wistar rats. In the present study, the potential relationship between drug-induced nephrotoxicity and the decrease in kidney calbindin-D 28kDa was investigated. Four groups of male Wistar rats were treated for 10 or 31 days with either the immunosuppressant CsA (50 mg/kg/day), FK-506 (5 mg/kg/day), rapamycin (5 mg/kg/day) or with the nonimmunosuppressive cyclosporine derivative 3'keto-[Bmt1]-[Val2]-CsA (SDZ PSC-833) (50 mg/kg/day), and the effects on calcium homeostasis, kidney histology and renal calbindin-D 28kDa were examined. Similar effects were found with CsA and FK-506; both drugs strongly reduced kidney calbindin-D 28kDa protein levels, increased urine calcium excretion, caused intratubular calcification, and induced basophilic tubules. In contrast, rapamycin and SDZ PSC-833 caused no decrease in renal calbindin-D 28kDa levels, no noticeable alterations in calcium metabolism, and no renal calcification. The results provide evidence for a link between decreased renal calbindin, increased calcium urine excretion, and intratubular kidney calcification. The present data show no correlation between the decrease in renal calbindin and the induction of basophilic tubules; however, it needs to be investigated if these apparently independent kidney effects may have a common origin upstream of calbindin expression.

Cyclosporine A decreases the protein level of the calcium-binding protein calbindin-D 28kDa in rat kidney.

Despite the widespread use of cyclosporine A (CsA), its mechanism of action and side effects are not yet completely understood. There exists a large body of evidence suggesting that disturbance of calcium homeostasis is a critical step in the cascade of cellular and molecular events induced by the drug. As recently shown in our laboratory by two-dimensional protein gel electrophoresis (2-DE) analysis of kidney homogenates, CsA induced numerous changes in several kidney proteins. One kidney protein in particular was shown to be strongly down-regulated by the drug. In this work we report the identification of the strongly decreased kidney protein as calbindin-D 28kDa, a vitamin D-dependent calcium-binding protein associated with calcium handling by cells. The assignment of the down-regulated protein spot is based on its internal amino acid sequence analysis and its specific reaction with a monoclonal antibody raised against calbindin-D 28kDa. In kidney homogenates of male Wistar rats treated with 50 mg/kg/d CsA for up to 28 days, calbindin levels were measured by ELISA and were shown to be continuously decreased with prolonged CsA treatment. To our knowledge, this is the first report describing the effect of CsA on kidney calbindin-D 28kDa protein levels. Further studies are needed to elucidate whether the CsA-mediated down-regulation of the calcium-binding protein calbindin-D 28kDa may be a critical factor for the renal adverse effects induced by this drug.

Protein variability in male and female Wistar rat liver proteins.

In the emerging field of mechanistic toxicology, growing attention is being paid to the interpretation of sex-related toxic responses. The Wistar rat outbred stock is a frequently used rodent for toxicity testing. Outbred strains, which display a relatively high degree of genetic variability between the individual animals are often used in risk assessment, as they are considered to best approximate the variability present in a human population. In this study two-dimensional (2-D) protein gel electrophoresis was applied to investigate the liver protein patterns of male and female Wistar rats and to search for (i) sex-related and (ii) interindividual qualitative and quantitative differences in protein expression. Among the sexes, six proteins were detected that were shown to be exclusively present in male rats and one that was present only in females. A male-specific protein was tentatively assigned to alpha 2u globulin. Seven protein spots showed statistically significant abundance changes (p < 0.001) between males and females, one was tentatively assigned to heme oxygenase 1 and another to the 23 kDa morphin-binding protein. Four sets of protein spots were detected that showed positional shifts in the individual patterns and are likely to represent polymorphic proteins inherent in the Wistar rat. These results form a valuable basis for future investigations of drug-induced changes in the male and female Wistar rat liver pattern.

Developmental expression of human microsomal epoxide hydrolase.

Microsomal epoxide hydrolase (mEH) is a critical biotransformation enzyme that catalyzes the hydrolysis of a large number of epoxide intermediates, which arise frequently from the oxidation of pharmaceutical and environmental compounds by the cytochrome P450 mixed function oxygenase system. The enzyme mEH has been implicated directly as a key determinant of certain chemically initiated cancers and developmental toxicities. To evaluate mEH expression in human tissues and provide a framework for assessing the relative risk in the fetus to potential developmental toxins, in the current study, the authors characterized mEH in human tissues as a function of gestational age. Analyses included enzymatic activity determinations, immunochemical quantitation of protein levels and RNA hybridization assays. With respect to activity, hepatic mEH enzymatic levels were strongly correlated with increasing gestational age (r = .82, P < .001). Similarly, mEH activity levels in the liver were highly correlated with protein contents (r = .93, P < .001). However, mEH enzymatic activity in the fetal lung did not exhibit similar concordance nor did measured RNA levels appear to correlate with enzymatic activity levels in fetal or adult tissue samples. Of the fetal tissues surveyed, the liver and adrenal glands exhibited the highest levels of detectable mEH RNA, followed by the lung and kidney. These data suggest that post-transcriptional regulatory pathways may be important in determining constitutive levels of mEH functional activity and that fetuses during early gestation may be uniquely sensitive to the presentation of epoxide-containing xenobiotics compared with fetuses at later stages of development.

Human microsomal epoxide hydrolase: genetic polymorphism and functional expression in vitro of amino acid variants.

Human microsomal epoxide hydrolase (mEH) is a biotransformation enzyme that metabolizes reactive epoxide intermediates to more water-soluble trans-dihydrodiol derivatives. We compared protein-coding sequences from six full-length human mEH DNA clones and assessed potential amino acid variation at seven positions. The prevalence of these variants was assessed in at least 37 unrelated individuals using polymerase chain reaction experiments. Only Tyr/His 113 (exon 3) and His/Arg 139 (exon 4) variants were observed. The genotype frequencies determined for residue 113 alleles indicate that this locus may not be in Hardy-Weinberg equilibrium, whereas frequencies observed for residue 139 alleles were similar to expected values. Nucleotide sequences coding for the variant amino acids were constructed in an mEH cDNA using site-directed mutagenesis, and each was expressed in vitro by transient transfection of COS-1 cells. Epoxide hydrolase mRNA level, catalytic activity, and immunoreactive protein were evaluated for each construct. The results of these analyses demonstrated relatively uniform levels of mEH RNA expression between the constructs. mEH enzymatic activity and immunoreactive protein were strongly correlated, indicating that mEH specific activity was similar for each variant. However, marked differences were noted in the relative amounts of immunoreactive protein and enzymatic activity resulting from the amino acid substitutions. These data suggest that common human mEH amino acid polymorphisms may alter enzymatic function, possibly by modifying protein stability.

Human peripheral lymphocytes as indicators of microsomal epoxide hydrolase activity in liver and lung.

In this study, we have applied an improved assay for the determination of microsomal epoxide hydrolase activity to assess enzymatic levels in human lung, liver, and blood lymphocytes. The assay is fluorescence-based and monitors the epoxide hydrolase-mediated conversion of (+/-)-benzo[a]pyrene-4,5-epoxide to (+/-)-trans-benzo[a]pyrene-4,5-dihydrodiol, using a high pressure liquid chromatography separation system. Approximately a 40-fold range in microsomal epoxide hydrolase activities was detected in blood lymphocytes collected from 70 individual donors. In 38 individuals who were sampled twice after a 3-month interval, the repeatability of an individual's lymphocyte epoxide hydrolase activity was highly correlated (r = 0.80, p < 0.02). In addition, within the same individual there appeared to be a strong correlation between lymphocyte and liver epoxide hydrolase activity (r = 0.92, p = 0.02), and some correlation between liver and lung activity (r = 0.58, p = 0.05). Activities were assessed in lymphocytes from a styrene-exposed worker population but no significant associations between blood concentrations of styrene and epoxide hydrolase activity levels were observed. Neither were any correlations detected in these workers between epoxide hydrolase activities and age, years on the job, alcohol consumption, sex, or smoking status. The results of our study suggest that blood lymphocytes are a useful sentinel cell for epoxide hydrolase activity determinations in individuals, as these measures are relatively stable over time and appear to reflect activity levels in other target organs.

Quantitative determination of porphyrins in rat and human urine and evaluation of urinary porphyrin profiles during mercury and lead exposures.

Measurement of urinary porphyrin excretion patterns (porphyrin profiles) is useful in the diagnosis and evaluation of diseases and disorders of porphyrin metabolism. However, experimental investigation of such disorders with rodent models has been hampered by the lack of an efficient procedure for the isolation and quantitative evaluation of porphyrins in rodent urine. This article describes an analytic procedure that overcomes the principal difficulties encountered with determination of porphyrins in rodent urine, including the loss of porphyrins during their isolation and interference of porphyrin fluorescence by contaminating materials. The procedure entails application of an acidified urine sample to a preconditioned C-18 preparatory column, preferential separation of essentially all potentially interfering contaminants by sequential phosphate-methanol elution, and selective isolation of porphyrins, which are then separated and quantitated by high-performance liquid chromatography and spectrofluorometric techniques. This method has been used to characterize urinary porphyrin excretion patterns in male rats and to define the distinctive changes in porphyrin profiles associated with prolonged exposure to porphyrinogenic metals. The porphyrin excretion patterns of male and female human subjects are also described. This method is applicable to the investigation of urinary porphyrin profile changes associated with exposure to a wide range of porphyrinogenic chemicals in both animals and human subjects.

Stimulation of porphyrinogen oxidation by mercuric ion. II. Promotion of oxidation from the interaction of mercuric ion, glutathione, and mitochondria-generated hydrogen peroxide.

Previous studies have shown that mercuric ion (Hg2+) reacts with GSH and H2O2 in vitro to form reactive species capable of oxidizing reduced porphyrins (porphyrinogens). This effect is independent of the presence of iron in the reaction mixture. The present studies demonstrate that Hg2+ and GSH can interact in biologically relevant concentrations with H2O2 generated by the mitochondrial electron transport chain to promote oxidation of porphyrinogens via comparable mechanisms. Mitochondria from rat liver or kidney readily oxidize uroporphyrinogen when H2O2 production is stimulated by the presence of a respiratory chain substrate (NADH, succinate) and an electron transport inhibitor (e.g., NaN3). Porphyrinogen oxidation by mitochondria is significantly increased by the addition of Hg2+ and GSH, in a molar ratio of approximately 3:5, to the reaction mixture. Stimulation of porphyrinogen oxidation in the presence of Hg2+ plus GSH increases proportionately with the concentration of mitochondrial protein in the reaction cuvettes but decreases with diminished H2O2 production by the electron transport chain. Studies with reactive oxidant scavengers suggest the participation of reactive oxygen species in Hg plus GSH stimulation of mitochondrial porphyrinogen oxidation. These findings support the hypothesis that Hg2+ and GSH interact with mitochondria-generated H2O2 to promote propagation of reactive oxidants or other free radical species, which, in turn, oxidize reduced porphyrins proximal to mitochondrial membranes. These results suggest a mechanistic explanation for the porphyrinogenic action of mercury compounds, as well as for the oxidative damage to target cell constituents associated with mercury exposure.

Stimulation of porphyrinogen oxidation by mercuric ion. I. Evidence of free radical formation in the presence of thiols and hydrogen peroxide.

The etiology of mercury-induced porphyrinuria was investigated by testing the hypothesis that mercuric ions (Hg2+) promote free radical-mediated oxidation of reduced porphyrins (porphyrinogens) by compromising the antioxidant potential of endogenous thiols, particularly GSH. Studies in vitro demonstrated that porphyrinogens (uroporphyrinogen and coproporphyrinogen) readily undergo H2O2-dependent oxidization in the presence of Fe3(+)-EDTA and that this action is attenuated by GSH at biologically relevant concentrations (0.5-10 mM). At low concentrations, Hg2+ complexes with GSH in a 1:2 molar ratio to decrease the antioxidant effect of GSH. However, at Hg2+ concentrations approaching saturation-complexation with available GSH, stimulation of porphyrinogen oxidation to 2 to 3 times that mediated by the H2O2/Fe3(+)-dependent system alone is observed. Stimulation of porphyrinogen oxidation by Hg2+ plus GSH increases in a dose-related manner with the concentration of H2O2 in the reaction mixture but is independent of the presence of iron. No porphyrinogen oxidation is observed in reaction mixtures containing H2O2 and either Hg2+ or GSH alone or when Hg+ is substituted for Hg2+. Studies with reactive oxidant scavengers and ESR spectroscopy suggest the participation of free radical species in Hg:GSH-mediated porphyrinogen oxidation. A mechanism involving ligand exchange between Hg2+ and GSH, which leads to formation of GS radicals and subsequent propagation of reactive oxygen-based radical species, is proposed. These studies support the view that Hg2+ both compromises the antioxidant potential of GSH and promotes formation of reactive species via thiol complexation. These findings suggest a mechanistic basis underlying the porphyrinogenic as well as tissue-damaging properties of mercuric ions.