Addition of high concentrations (>1 mM) of inorganic phosphate (P i ) or arsenate to Ca 2؉ -loaded mitochondria was followed by increased rates of H 2 O 2 production, membrane lipid peroxidation, and swelling. Mitochondrial swelling was only partially prevented either by butylhydroxytoluene, an inhibitor of lipid peroxidation, or cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. This swelling was totally prevented by the simultaneous presence of these compounds. At lower P i concentrations (1 mM), mitochondrial swelling is reversible and prevented by cyclosporin A, but not by butylhydroxytoluene. In any case (low or high phosphate concentration) exogenous catalase prevented mitochondrial swelling, suggesting that reactive oxygen species (ROS) participate in these mechanisms. Altogether, the data suggest that, at low P i concentrations, membrane permeabilization is reversible and mediated by opening of the mitochondrial permeability transition pore, whereas at high P i concentrations, membrane permeabilization is irreversible because lipid peroxidation also takes place. Under these conditions, lipid peroxidation is strongly inhibited by sorbate, a putative quencher of triplet carbonyl species. This suggests that high P i or arsenate concentrations stimulate propagation of the peroxidative reactions initiated by mitochondrial-generated ROS because these anions are able to catalyze C n -aldehyde tautomerization producing enols, which can be oxidized by hemeproteins to yield the lower C n ؊ 1 -aldehyde in the triplet state. This proposition was also supported by experiments using a model system consisting of phosphatidylcholine/dicethylphosphate liposomes and the triplet acetone-generating system isobutanal/horseradish peroxidase, where phosphate and Ca 2؉ cooperate to increase the yield of thiobarbituric acid-reactive substances.The decrease in ATP levels that occurs under pathological conditions such as prolonged anoxia or ischemia/reperfusion results in inorganic phosphate (P i ) accumulation and alterations in intracellular Ca 2ϩ homeostasis (1-7). It is argued that high P i concentrations mimic the metabolic conditions prevalent during ischemia and that, at concentrations higher than 10 mM, P i inhibits mitochondrial oxidative phosphorylation (8). Indeed, it is well known (see Refs. 9 and 10, and references therein) that accumulation of Ca 2ϩ and P i by mitochondria results in increased permeability of the inner mitochondrial membrane. This is proposed to be a key step in the pathogenesis of cell injury that occurs during ischemia and reperfusion (8 -11). Different mechanisms such as lipid peroxidation, phospholipid hydrolysis by phospholipase A 2 , or opening of the mitochondrial permeability transition pore (MTP) 1 have been proposed to take place under these conditions (9 -14). In this regard, P i concentration, which is reported to increase up to 20 mM in cells exposed to prolonged anoxia (6), has a profound effect in the rate, extent, and nature of mitochondrial membrane altera...

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1. The enolamine form of 5-aminolaevulinic acid (ALA), a haem precursor that accumulates in lead poisoning and in acute intermittent porphyria (AIP), undergoes fast autoxidation at slightly alkaline pH with concomitant generation of reactive oxygen species. 2. The transmembrane potential, Ca2+ ion fluxes and state-4 respiratory rate, of isolated rat liver mitochondria are severely affected by mM addition of ALA; the toxic role of ALA-produced oxygen radicals was demonstrated by use of appropriate scavengers. 3. Induction of superoxide dismutase biosynthesis in lead-exposed workers, in AIP carriers and in ALA-treated rats, is viewed as a protective response against oxygen radical toxicity. 4. 5-Aminolaevulinic acid-generated oxygen radicals, together with Pb-stimulated Fe-dependent lipid peroxidation, might be involved in the aetiology of the neuropsychiatric manifestations of both plumbism and acute intermittent porphyria.

The larval click-beetle Pyrearinus termitilluminans elicits the phenomenon of luminous termite mounds in the central-west region of Brazil. The bioluminescence (BL) spectrum of this larva (lambda max = 534 nm) is one of the most blue-shifted reported among known luminescent Coleoptera. We have isolated mRNA from larval thoracic lanterns and constructed a cDNA library into a lambda ZAP II vector. An expression library was obtained after excision of the pBluescript plasmid. This library was screened by photodetection and one clone that emitted green BL (lambda max = 538 nm) was isolated. The 2.2 kb cDNA insert includes a 543 residue open reading frame showing 82% homology with the luciferase isoenzymes of Pyrophorus plagiophthalamus (Coleoptera: Elateridae). As expected, the region between residues 223 and 247 that contains the putative active site for BL color determination showed a higher degree of homology among click-beetle luciferases that elicit closer BL colors. The in vitro BL spectrum of recombinant P. termitilluminans luciferase also peaks at 538 nm and, as in the case of native enzyme, does not show any bathochromic shift upon decreasing the pH.

Lead-induced neurotoxicity acquired by low-level long-term exposure has special relevance for children. A plethora of recent reports has demonstrated a direct link between low-level lead exposure and deficits in the neurobehavioral-cognitive performance manifested from childhood through adolescence. In many studies, aggressiveness and delinquency have also been suggested as symptoms of lead poisoning. Several environmental, occupational and domestic sources of contaminant lead and consequent health risks are largely identified and understood, but the occurrences of lead poisoning remain numerous. There is an urgent need for public health policies to prevent lead poisoning so as to reduce individual and societal damages and losses. In this paper we describe unsuspected sources of contaminant lead, discuss the economic losses and urban violence possibly associated with lead contamination and review the molecular basis of lead-induced neurotoxicity, emphasizing its effects on the social behavior, delinquency and IQ of children and adolescents.

Porphyrias are inherited and acquired diseases of erythroid or hepatic origin, in which there are defects in specific enzymes of the heme biosynthetic pathway. In patients with intermittent acute porphyria and lead poisoning the erythrocytic activities of superoxide dismutase and glutathione peroxidase are reported to be increased. Our studies demonstrated that d-aminolevulinic acid, a heme precursor accumulated in both diseases, undergoes enolization at pH less than 7.0 before it autoxidizes. The autoxidation of d-aminolevulinic acid, in the presence or absence of oxyhemoglobin has been proposed as a source of oxy and carbon-centred radicals in the cells of intermittent acute porphyria and saturnism carriers. Thus, the increased levels of antioxidant enzymes can be viewed as an intracellular response against the deleterious effects of these extremely reactive species.