Defining thresholds in occupational and environmental toxicology.

When a chemical causes a defined form of toxicity, the threshold is the maximum exposure when this toxicity does not occur. It is an operational parameter and is limited in its interpretation and applicability. The aim of this paper is to consider biological parameters which influence exposure-response relationships. Biomonitoring of dose and effects has much potential for defining thresholds in human exposure; extension of their use in experimental studies on new compounds should help predictions to thresholds for human exposure. Intoxication initiated by both reversible and covalent interactions with targets are discussed and, as exposure is reduced and the time of exposure extended, changes in the shape of the dose-response curves examined for acute and delayed neuropathy (axonopathy) and for carcinogenesis.

The esterases: perspectives and problems.

Many proteins capable of hydrolysing esters are present in biological material of all kinds (microorganisms, plants, invertebrates and vertebrates). Some serve, as indicated by their substrate specificity and distribution within organisms, a defined biological function. However for most esterases a rather general substrate specificity is found indicating that they may have a broad biological function. Their properties will be briefly reviewed with particular emphasis on inhibitors. The mechanism of hydrolysis of esters by many carboxylesterases (B-esterases) is well established largely due to the reaction of OP compounds with their catalytic centre. For others, such as enzymes hydrolysing (i) OP compounds and/or (ii) carboxyl esters which are not inhibited by a time and temperature dependent reaction by OP compounds, reaction mechanisms are still conjecture. The purpose of this presentation is to explore similarities and differences between the esterases and to discuss possible routes for progress in the A-esterase group.

The toxic oil syndrome (TOS, 1981): from the disease towards a toxicological understanding of its chemical aetiology and mechanism.

In Spain early in May 1981, 20,000 people became ill with a severe acute respiratory illness. The eosinophilia and subsequent myalgia, scleroderma and muscle wasting indicated a unique disease entity. Epidemiological evidence linked the disease with the consumption of oils containing "refined" aniline denatured rape seed oil. Ten years after the explosive appearance of this disease (approximately 350 deaths and over 1000 in the chronic phase) the clinical and pathological description is now well established. The aetiological agent(s) in the food oil are unknown and the mechanism(s) of pathogenesis are uncertain. There is no experimental animal model. A new disease, Eosinophilia Myalgia Syndrome (EMS) which appeared late in 1989 in the USA, is due to the consumption of impure 1-tryptophan. There may be similarities between the diseases and the aetiological agents for TOS and EMS: possibilities for future research will be discussed. Underlying the time lag for solution of this problem is a lack of knowledge of the basic biology involved.

The advisory subgroup in toxicology of the european medical research councils.

The European Medical Research Councils set up an Advisory Group in Toxicology which met from 1975 to 1988. Since encouragement of cross discipline research is still difficult, a resumé is presented of the procedures developed to encourage interdisciplinary research in toxicology in Europe. A programme of grants in toxicology for collaborative research between European countries was begun in 1981 under the auspices of the European Science Foundation. Resulting from the final meeting of AST in Milan, the need for the development of links between epidemiology and molecular aspects of toxicology and for new approaches in eco-toxicology are briefly discussed.

Studies on the metabolism of the pneumotoxin O,S,S-trimethyl phosphorodithioate--I. Lung and liver microsomes.

The metabolism of O,S,S-trimethyl phosphorodithioate (OSSMe), a pneumotoxic impurity in some organophosphorus insecticides, was investigated in rat lung and liver microsomal preparations, using OSSMe labelled with 3H or 14C on one of its thiolo-methyl (CH3S-) groups. Production of O,S-dimethyl phosphorothioate (OSMeO-) and binding of radioactivity to protein were NADPH-dependent and were shown to be, at least partly, cytochrome P-450-dependent processes in both lung and liver microsomes. Incubation with reduced glutathione prevented the binding of radioactivity without affecting OSMeO- production. The Km for the conversion of OSSMe to OSMeO- was 15-fold lower in lung (0.30 +/- 0.07 mM) than in liver (4.63 +/- 2.42 mM) microsomes. These results show that cytochrome P-450-dependent mixed-function oxidase is implicated in at least part of the metabolic activation of OSSMe, and suggest that the pulmonary isozyme(s) are more active at metabolizing OSSMe than hepatic isozymes. It is speculated, on the basis of literature data on other sulphur-containing chemicals, that the metabolic activation of OSSMe involves oxidation of a thiolo-sulphur, with subsequent formation of CH3-S-S-protein disulphides.

Studies on the metabolism of the pneumotoxin O,S,S-trimethyl phosphorodithioate--II. Lung and liver slices.

The metabolism of O,S,S-trimethyl phosphorodithioate (OSSMe), a pneumotoxic impurity in some organophosphorus insecticides, was investigated by incubating rat lung and liver slices with 1 mM OSSMe, labelled with 3H or 14C on one of its thiolo-methyl (CH3S-) groups. Protein bound radioactivity was higher in lung slices than in liver slices. In lung slices the predominant diester produced was O,S-dimethyl phosphorothioate (OSMeO-), whereas in liver slices it was S,S-dimethyl phosphorodithioate (SSMeO-). Other studies had shown binding of radioactivity and OSMeO- production to be cytochrome P-450-dependent processes in microsomes and SSMeO- production to result from the action of cytosolic glutathione-S-transferase on OSSMe. Preincubation of slices with 10(-5) M paraoxon did not influence the amount of protein-bound radioactivity, suggesting that binding of radioactivity did not simply result from protein phosphorylation. Pretreatments of the rats with O,O,O-trimethyl phosphorothioate [OOOMe(S) 0.5, 2.5 and 12.5 mg/kg p.o.], with p-xylene (1 g/kg, i.p.) or with bromophos (5.3 mg/kg, i.p.) which all protect against the lung toxicity of OSSMe probably by inhibiting pulmonary mixed-function oxidase, also led to significant decreases in both protein binding of radioactivity and OSMeO- production in lung slices, but not in liver slices. These results show that tissue slices are a convenient system for investigating xenobiotic metabolism in the lung and they suggest that the susceptibility of the lung to OSSMe probably results from a relatively high rate of activation, coupled with a relatively low rate of metabolism by non-toxic pathways and/or removal of reactive metabolites in some lung cells, possibly the alveolar type I cells.

Putrescine and 5-hydroxytryptamine accumulation in rat lung slices: cellular localization and responses to cell-specific lung injury.

The cellular localization of putrescine (1,4-diaminobutane) and 5-hydroxytryptamine (5HT) following the accumulation of tritium-labeled putrescine (2.5 microM) or 5HT (0.5 microM) into rat lung slices was determined by autoradiography at the light microscope level. Putrescine labeling was found to occur in type II alveolar epithelial cells and in branchiolar nonciliated (Clara) cells, and possibly also in type I alveolar epithelial cells. The pattern of 5HT labeling was clearly different from that with putrescine, since the parenchyma was diffusely labeled with no preferential location in type II cells, but with strong labeling of the endothelium of large vessels and also the pleural mesothelium. The apparent kinetic parameters for the tissue uptake of [3H]putrescine (2.5 to 80 microM) and [14C]5HT (0.5 to 16 microM; both being simultaneously present in a 5 to 1 molar ratio) were studied in lung slices from normal rats and rats pretreated with O,S,S-trimethyl phosphorodithioate (OSSMe, 11 to 95 mg/kg, po), with paraquat (20 mg/kg, ip), or with alpha-naphthylthiourea (ANTU, 5 or 10 mg/kg, ip). OSSMe and paraquat were used as models for pulmonary epithelium-damaging agents, and ANTU was taken as a model for a pulmonary endothelium-damaging agent. The Vmax for the uptake of 5HT was significantly increased (without change in Km) following treatment with OSSMe and paraquat. Following ANTU treatment the Vmax for the uptake of 5HT was unchanged (5 mg/kg) or increased (10 mg/kg, Km also increased). These results indicate that in lung slices the response to lung injury may be associated with an increased accumulation of 5HT. The Vmax for the uptake of putrescine was significantly decreased (without change in Km) following treatment with OSSMe and paraquat. Following ANTU treatment the Vmax for the uptake of putrescine was unchanged (5 mg/kg) or decreased (10 mg/kg, no change in Km). These results suggest that a decreased putrescine uptake is a sensitive index of pulmonary epithelial damage.

The interaction between phosphorothionate insecticides, pneumotoxic trialkyl phosphorothiolates and effects on lung 7-ethoxycoumarin O-deethylase activity.

A number of phosphorothionate (P = S) insecticides, including bromophos and fenitrothion, prevent trialkyl phosphorothiolate (P = O)-induced lung toxicity and the resulting increase in lung weight normally observed at 3 days in the rat. Measurement of 7-ethoxycoumarin O-deethylase (7-EC) activity after both phosphorothionate and phosphorothiolate dosing revealed differing patterns of loss of enzyme activity. Depletion of 7-EC activity by phosphorothionates was maximal between 2 and 10 h after dosing, with recovery between 24 and 72 h. Phosphorothiolates, however, appear to cause two phases of loss of 7-EC activity, an initial fall of approximately 30% observed at 2 h and a secondary fall, maximal on day 3, with loss of 97% of activity, apparently associated with the pathological changes in the lung. It is suggested that oxidative metabolism of phosphorothionates known to occur at the P = S moiety, with suicidal loss of P-450, may then prevent oxidative activation of an S-methyl on the phosphorothiolates, the most likely site for production of a reactive intermediate capable of damaging the lung. Lung 7-EC in rat is sensitive to concentrations of the phosphorothionates bromophos and fenitrothion at 5-25 times less than those causing loss of liver 7-EC activity and at doses 125-600 times less than their LD50s. If repeated in man this may have implications for personnel occupationally exposed to these compounds.

The toxicity and neuropathology of dimethylethyltin and methyldiethyltin in rats.

Triethyltin causes an increase in brain water with vacuolation of myelin sheaths, whereas trimethyltin is selectively damaging to neurons, especially of the hippocampal formations, causing chromatolysis, accumulation of cytoplasmic dense bodies and often cell death. The effects on rats of the analogues, dimethylethyltin and methyldiethyltin (oral LD50 14 mg/kg and 7.5-10.0 mg/kg respectively) are now reported. The dimethylethyl compound produces functional changes resembling those caused by trimethyltin, while the methyldiethyl compound causes responses similar to those produced by triethyltin. Structurally, however, the dimethylethyl compound, while producing marked nerve cell changes of the trimethyltin type also causes moderate vacuolation of myelin sheaths. By contrast, methyldiethyltin causes marked vacuolation of myelin sheaths of the triethyltin type and relatively minor neuronal changes of the trimethyltin type. These findings are discussed in terms of the structure-activity relationships of trialkyltin compounds.

Morphological and biochemical correlates of chemical induced injury in the lung. A discussion.

We have reviewed some of the factors which contribute to lung damage by various toxicants. These include disposition of the chemical, its metabolism, individual cell type susceptibility and the potential for the tissue to repair. We have discussed the use of biochemical parameters to measure the functional activity of individual cell types in order to predict the damage to specific cell types and concluded that careful morphological analysis of lung tissue is likely to provide a more sensitive and informative measure of specific cell type injury. However, in order to investigate the mechanism of toxicity of pulmonary toxicants it is essential to establish the primary biochemical event that leads to cell damage and morphological change. The importance of separating the relevant biochemical change(s) from the cascade of biochemical events associated with dead and dying cells and the reparative response of the lung is emphasised.

Some aspects of the toxicology of trimethyl and triethyl phosphorothioates.

The pharmacokinetics (disposal curves) of trimethyl and triethyl phosphorothioates have been determined. The concentrations to which the lung has been exposed at the LD50 dose of different chemical structures have been compared with the dose administered to the animal; the variation of LD50 of different chemical structures is little reduced. The in vitro kinetics of the reaction of O,S,S-trimethyl phosphorodithioate or O,O,S-triethyl phosphorothioate with plasma cholinesterase and carboxylesterase and brain acetylcholinesterase have been determined. The relation between inhibition and circulating concentrations in vivo have been examined. Changes in Clara cells reported by others seem to be physiological rather than pathological. O,S,S-Trimethyl phosphorodithioate is metabolised by rat lung and liver slices and microsomes. From these studies and the effect of various pretreatments of the rats on toxicity to the lung, it is probable that the proximal toxin is produced in the lung by oxidative attack on the alkylthio moeity of the compounds.

Trialkyl phosphorothioates and glutathione S-transferases.

Using a rat liver cytosol source of enzyme trialkyl phosphorothioates have been shown to be substrates of glutathione S-transferases. Using OSS-trimethyl phosphorodithioate (OSS-Me(O] and OOS-trimethyl phosphorothioate (OOS-Me(O] the methyl transferred to the sulphydryl of glutathione is that attached to phosphorus via an oxygen atom. Fractionation of liver cytosol has shown that although the bulk activity is due to the three isozymes (1-1; 3-4; 1.2), OSS-Me(O) is a general substrate for glutathione S-transferases. The specific activity is low compared with the substrates 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene.

Evolution of the intracellular changes in neurons caused by trimethyltin.

Rats have been given a single dose of trimethyltin (10 mg/kg) and the intracellular events have been followed particularly in hippocampus, cerebral cortex, cerebellum and spinal ganglion cells. The earliest change visible occurs 12 h after this dose and is found to be dense membrane-bound bodies, probably derived from branching tubulo-vesicular smooth endoplasmic reticulum formations. These occur in close connection with rought endoplasmic reticulum and polyribosomes and appear also to have some association with the Golgi complex. At 24 h there is a general vacuolation of Golgi cisterns and SER membranes, and the membrane-bound dense body formation is greatly increased. SER abnormalities are particularly conspicuous in Purkinje cells. In spinal ganglion cells, while vacuolation of Golgi cisterns is intense, dense bodies are inconspicuous and are replaced by increased autophagosomes, often of great complexity. By 48 h vacuolation of Golgi cisterns has waned, but accumulation of dense bodies and secondary lysosomes has steadily increased. In spinal ganglion cells autophagosomes only are increased as the Golgi vacuolation declines. At later times steady increases of lysosomal dense bodies is seen generally accompanied in hippocampal pyramidal cells and dentate fascia cells by abundant cell death. The suggestion is put forward that the Golgi complex may be the seat of the critical metabolic lesion and disturbances to protein transfer and protein synthesis follow. No explanation for the selective loss of hippocampal h1-5 (CA1-CA4 except Sommer's sector) pyramidal cells and of small dentate fascia neurons can be derived from these conclusions.

The binding of triethyllead to rat and cat hemoglobin.

Equilibrium dialysis experiments were carried out to determine the affinity constant and stoichiometry of the binding of triethyllead to rat and cat hemoglobin. The affinity constants were 4 X 10(5) M-1 and 4.5 X 10(5) M-1 for rat and cat hemoglobin, respectively, and 2 mol triethyllead combined with 1 mol hemoglobin. The previous report of a stoichiometry of 3 could not be confirmed (Byington et al., 1980).