Comparative toxicity of alkyl-1,4-naphthoquinones in rats: relationship to free radical production in vitro.

2-Methyl-1,4-naphthoquinone causes haemolysis in vivo. This toxic effect is believed to result from oxidative damage to erythrocytes by "active oxygen" species formed via one-electron reduction of the naphthoquinone by oxyhaemoglobin. In the present investigation, seven 2-alkyl-1,4-naphtoquinones have been studied with regard to their haemolytic activity in rats, their ability to cause oxidative damage in erythrocytes in vitro, and their reactivity toward oxyhaemoglobin. A close correlation was observed between the in vivo and in vitro parameters, suggesting that the proposed mechanism of toxicity of 2-methyl-1,4-naphthoquinone is correct and is also applicable to other alkylnaphthoquinones.

Generation of superoxide radical and hydrogen peroxide by 2,3,6-triaminopyridine, a metabolite of the urinary tract analgesic phenazopyridine.

2,3,6-Triaminopyridine, a metabolite of the widely-used drug phenazopyridine, has been shown to autoxidize at neutral pH, generating superoxide radical and hydrogen peroxide. Hydrogen peroxide was also detected in erythrocytes exposed to this substance, and these cells suffered oxidative damage, as reflected by methaemoglobin formation and glutathione depletion. The in vitro effects of 2,3,6-triaminopyridine are closely similar to those of the structurally-related compound, 1,2,4-triaminobenzene. The latter substance is known to be highly toxic in vivo by mechanisms which may involve free radical production and oxidative stress. It is possible, therefore, that triaminopyridine may be similarly toxic in animals and that this metabolite could be responsible for some of the harmful side-effects associated with phenazopyridine use.

Muscle necrosis by N-methylated p-phenylenediamines in rats: structure-activity relationships and correlation with free-radical production in vitro.

Certain derivatives of p-phenylenediamine have been shown to cause necrosis of cardiac and skeletal muscle in rats; in vitro, such compounds are known to autoxidize to the corresponding radical cations, with concomitant formation of oxygen free-radicals. In the present study, the autoxidation rates of p-phenylenediamine and its N-methyl, dimethyl, trimethyl and tetramethyl derivatives have been determined and compared with the severity of the muscle necrosis induced by each of these compounds in rats. A close correlation was observed between autoxidation rate in vitro and toxicity in vivo, suggesting that free-radical species may be involved in the initiation of the muscle damage caused by these substances.

Structure-activity relationships in the myotoxicity of ring-methylated p-phenylenediamines in rats and correlation with autoxidation rates in vitro.

A number of N-methylated p-phenylenediamines are known to cause necrosis of skeletal and cardiac muscle in rats. The severity of the muscle damage induced by these compounds in vivo was found to be directly proportional to their autoxidation rates in vitro, suggesting that reactive species formed during oxidation may be involved in the initiation of this toxic effect. In the present study, the in vitro oxidation rates and in vivo toxicities of a number of ring-methylated p-phenylenediamines have been evaluated. 2,3,5,6-Tetramethyl p-phenylenediamine readily autoxidized at neutral pH. Hydrogen peroxide was formed in this reaction, while oxidation in the presence of glutathione or reduced pyridine nucleotides led to the production of both hydrogen peroxide and superoxide radical. Less highly methylated derivatives oxidized more slowly, with rates decreasing in the order 2,3,5,6-tetramethyl greater than 2,5-dimethyl greater than 2,6-dimethyl greater than 2-methyl. All these compounds were myotoxic in rats, with damage being largely confined to skeletal muscle. Toxicity was again proportional to oxidation rate. Myotoxicity appears to be a general property of certain substituted p-phenylenediamines and the structure-activity relationships identified may permit an estimate to be made of the potential toxicity of other compounds of this type.

In vitro and in vivo mutagenicity studies on sporidesmin, the toxin associated with facial eczema in ruminants.

Sporidesmin, a fungal toxin with widespread distribution within New Zealand, is thought to exert toxic effects through oxidative damage. The purified chemical was tested for its ability to cause point mutations in four strains of Salmonella typhimurium (TA98, TA100, TA102 and TA1537), in the presence and absence of exogenous metabolic activation. Although toxic effects were seen at concentrations exceeding 400 mu gl/plate, there were no significant increases in revertant colonies. In strain TA102, these results were not modified by the presence of glutathione. In AA8 Chinese hamster cells, sporidesmin acted as a potent clastogen, causing chromosomal breaks at concentrations as low as 3 ng/ml, where there was very little reduction in cell viability. Effects were primarily at the chromatid level, but some chromosomal events were also seen. Following low doses, the most common events were chromatid deletions and induction of double minute chromosomes. Interchange events occurred at concentrations of 10 ng/ml and above. The most common of these events was an incomplete chromatid interchange, although some examples of complete chromatid and chromosomal interchange were seen. These in vitro experiments were subsequently extended to an in vivo study of sporidesmin-induced lymphocytic micronuclei (MN) in sheep. In a double blind experiment, 5 sheep were treated with a single high dose of sporidesmin. Blood samples were taken from these, and from 5 untreated sheep, at various intervals before and after treatment. Peripheral blood lymphocytes cultures were harvested and scored for MN in cytokinesis-blocked cells, as a measure of clastogenic activity of sporidesmin in vivo. Following decoding, statistical analysis of the data revealed no significant differences between the MN levels in peripheral blood lymphocytes of sporidesmin-treated and untreated sheep. Although the possibility still exists that clastogenic effects could occur in other species, the data indicate that sporidesmin is not a clastogen in sheep, even though this species is highly susceptible to the toxic effects of sporidesmin.

Steric effects on the haemolytic activity of aromatic disulphides in rats.

Certain derivatives of diphenyl disulphide are known to cause haemolytic anaemia in rats, by a mechanism possibly involving intra-erythrocytic redox cycling with concomitant generation of 'active oxygen' species. In ring-substituted diphenyl disulphide derivatives, electronic effects of substituents have been shown markedly to affect the rate of 'active oxygen' production in vitro and toxicity in vivo. In the present study, the influence of steric effects of substituents on these parameters has been investigated. The severity of the haemolysis induced in groups of seven rats by oral dosing with 4,4'-dimethoxydiphenyl disulphide and 4,4'-dimethyldiphenyl disulphide at doses of 500 mumol/kg/day for 6 days was greater than that of the 2,2' isomers and the haemolytic activity of a series of 2,2'-dialkyl derivatives decreased with increasing size of the alkyl group. In vitro, the haematin-catalysed oxidation rates and the rates of redox cycling of the corresponding thiols in the presence of glutathione were similarly influenced by steric hindrance. The structure-activity relationships identified in the present investigation, together with knowledge of the electronic effects of substituents, should permit accurate prediction of the toxicity of new or untested aromatic thiols and disulphides.

Haemolytic activity and nephrotoxicity of 2-hydroxy-1,4-naphthoquinone in rats.

The short-term toxicity of 2-hydroxy-1,4-naphthoquinone (lawsone) and 2-methyl-1,4-naphthoquinone (menadione) has been compared in rats. 2-Methyl-1,4-naphthoquinone has been shown previously to cause haemolytic anaemia in animals, and this was confirmed in the present experiment. 2-Hydroxyl-1,4-naphthoquinone was found also to cause haemolysis, in a dose-dependent manner, as reflected by decreased blood packed cell volumes and haemoglobin levels and by histopathological changes in spleen, liver and kidney. With both naphthoquinones, the haemolysis was of the oxidative type, characterized by the presence of Heinz bodies within erythrocytes. Haemolysis was the only toxic change identified in rats dosed with 2-methyl-1,4-naphthoquinone. In contrast, 2-hydroxyl-1,4-naphthoquinone was not only a haemolytic agent but also a nephrotoxin, causing renal enlargement, elevated plasma levels of urea and creatinine and histologically-identified tubular necrosis, largely confined to the distal segment of the proximal convoluted tubules. The relationship between the in vivo toxic effects of these naphthoquinones and previously-reported data on their in vitro cytotoxic action is discussed.

A zinc-containing intraruminal device for facial eczema control in lambs.

A zinc-containing intraruminal device has been developed for protecting lambs against facial eczema. The rate of release of zinc from the device has been optimised, and its safety in use established. Under both experimental and farm conditions, the device gave excellent protection against the liver injury associated with facial eczema. The device relies upon erosion for release of zinc, and disappears completely when its charge of zinc has been released, leaving no metal or plastic residue in the rumen. This device has the potential to greatly ameliorate the problem of facial eczema in New Zealand.