Theoretical mechanistic basis of oxidants of methaemoglobin formation.

The mechanism of oxidation or reduction using the electron method was investigated for (I) aniline; (II) nitrobenzene; (III) nitrate; (IV) sulphanilamide; (V) hydrogen peroxide; (VI) hydroxyl free radical; (VII) ferricyanide; (VIII) acetylphenylhydrazine; (IX) nitrite; (X) chlorate and (XI) hydroxylamine respectively. Substances (II), (III), (V), (VI), (VII), (IX), (X) and (XI) evolved as oxidants, with (II), nitrobenzene and (X), chlorate as the most powerful oxidants (number of moles of HbFe(2+)(haemoglobin) of 6 reacting with 1.0 mole of the substance). Substances (I), (IV) and (VII) evolved as reductants of equal reducing power (number of moles of HbFe(3+)(methaemoglobin) of 4 reacting with 1.0 mole of the substance). Using the following equations, the impact of oxidants and reductants on glutathione (GSH) peroxidase, glutathione (GSSC) reductase and NADHmetHb reductase respectively on methaemoglobinaemia generation was investigated. [Equation in text]. Redox potential change (DeltaE' (o)) of 1.77, -1.77 and 1.86 volt and free energy change (DeltaG(o)') of -81, 81 and -85.8 kcal/mol were calculated for GSH peroxidase, GSSG reductase and NADHmetHb reductase systems respectively. In sustained methaemoglobinaemia, these mechanisms predict low levels of NADHmetHb reductase and glutathione peroxidase respectively, but high levels of glutathione reductase in red blood cells on exposure to oxidants. The significance of these mechanisms was investigated in cord blood, neonatal, adult red blood cells and other biological systems. It was concluded that any reaction with a positive DeltaE(o)' and negative DeltaG(o)' with the Fe(3+): Fe(2+)couple will indicate methaemoglobin oxidizing power. The effects on red blood cells and white blood cells were manifested in the biochemical toxicology of nitroso (PhN = 0), arylamine glucuronide (PhNHG) and arene imine respectively.

Mechanistic biotransformations of some amphetamine drugs.

Amphetamine, fenfluramine and benzphetamine were the drugs investigated for the isolation of toxic metabolites using the biochemical mechanism of cytochrome P-450 monooxygenase mediated reaction. NH3 derived from amphetamine should be innocuous unless the in vivo ammonia detoxifying mechanism is overwhelmed thus culminating in ammonia intoxication in cerebral tissues with consequent concomitant convulsion. +CF3 electrophile derived from fenfluramine is potentially reactive with nucleophiles of proteins, carbohydrates, lipids, DNA and RNA. The derivation of .CF3 was discussed. Methylbenzylamine was derived from benzphetamine. This, in the nitrosating environment of the gastrointestinal tract, could yield the carcinogenic methylbenzylnitrosamine.

Detachment of ribosomes of rough endoplasmic reticulum by NADPH and reduced glutathione (GSH).

NADPH, an endogenous, naturally occurring compound was found to be positive in the stripping of ribosomes from rough endoplasmic reticulum (RER) test (degranulation) for carcinogens using the hepatocellular postmitochondrial (PMS) system. However, glutathione (GSH) another naturally occurring compound was negative in the hepatocellular PMS system in contrast to GSH which was previously reported as being positive in the mutagenicity test using kidney PMS but negative using liver PMS. Akintonwa has classified degranulation into three types, viz, DIA, DIB, and DII (D.A.A. Akintonwa, Ecotoxicol. Environ. Saf. 9, 53-63 (1985]. Most carcinogens fall into DIA and DIB types. NADPH was of DIA type in the mouse and of DII in the female rat PMS systems. NADPH was consistently negative in the male rat PMS system.

Mechanism of RNA redistribution of the 17,000g postmitochondrial supernatant after incubation at 37 degrees C and its impact on other related biochemical investigations.

Evidence for stripping of ribosomes from RER in the 17,000g PMS of the liver of the cancer patient was obtained in the 1.35 M region which is the region between the SER and RER. RNA/protein ratios for the SER, 1.35 M region, and RER of 0.001, 0.083, and 0.235, respectively, for this liver are consistent with the degranulation of RER compared with RNA/protein ratios for SER and RER from normal livers of 0.025 +/- 0.003, and 0.35 + 0.030, respectively. A RNA/protein ratio of 0.235 was obtained for the RER region of the cancer patient. The EM revealed that the region of the RER in the cancer liver was ribosomal and not at all RER. This ribosomal material is reminiscent of the ribosomes banding in the 1.35- to 2 M domain of the RER isolated from the reconstitution experiments (SER + polyribosomes + supernatant). It was suggested that the ribosomal material isolated from the cancer liver could therefore be indicative of polyribosomal lesion or degradation. The 0.25-1.35 M interface (SER), 1.35 M region, and 1.35-2 M interface (RER) characteristics are therefore exploitable for diagnostic potential and further understanding of the molecular basis of cancer.

High-pressure liquid chromatography separation of phenobarbitone, thiourea, and thioacetamide of toxicological interest.

It was possible, by reverse-phase high-pressure liquid chromatography (HPLC) to separate thiourea, thioacetamide, and phenobarbitone with retention times of 1.4, 1.7, and 4.7 min, respectively. Thiourea and thioacetamide are known carcinogens. Substances such as phenobarbitone, thiourea, and thioacetamide, which may be encountered in our environment, can now be identified by this technique. It is also recommended that this HPLC technique should be used routinely in the screening of the purity of substances which are being subjected to toxicity, carcinogenicity, mutagenicity, and teratogenicity evaluation. A mixture of impurities in the sodium phenobarbitone was removed in the synthesis of the 5-ethyl-5-phenyl-barbituric acid (phenobarbitone). These substances which were not thiourea or thioacetamide could have been responsible for the positive result in the in vitro degranulation test for carcinogens with sodium phenobarbitone.

Studies on some biological differences between the chiral forms of methyl-1-naphthyl-p-nitrophenyl phosphorothionate.

A general degranulation test for carcinogens using the postmitochondrial supernatant (PMS) was based on the consistency of the stripping of ribosomes from rough endoplasmic reticulum. The degranulation phenomenon has been classified into three types, which are D1A, D1B and D11. This classification which is new has been introduced to probe more into the mechanistic aspects of degranulation. Aflatoxin B1 dimethylnitrosamine, 2-acetylaminofluorene, and 4-dimethylaminobenzene were carcinogens, evaluated in the PMS system. Reduced glutathione (GSH), oxidized glutathione (GSSG), sodium chloride, potassium chloride, lithium chloride, lithium carbonate, and ascorbic acid which are noncarcinogens and the (+) and (-) chiral forms of methyl-1-naphthyl-p-nitrophenyl phosphorothionate were also evaluated in the PMS system. Lithium carbonate is teratogenic in rats. Similar findings have also been reported in mice, and lithium ion has been confirmed to be teratogenic in mice. This biological phenomenon of the D11 type could well evolve as a teratogenic effect and not a carcinogenic effect.

The derivation of nitrosamines from some therapeutic amines in the human environment.

Five therapeutic drugs which are secondary and tertiary amines were investigated by reaction mechanisms for the derivation of nitrosamines in the human environments. These drugs are chlorpromazine (tranquilizer), methadone (analgesic), chloroquine (antimalarial), primaquine (antimalarial), and phenacetin (analgesic). Phenacetin is an N-acetylated secondary amine; chloroquine and primaquine are secondary amines; methadone and chlorpromazine are tertiary amines; and chloroquine is also a tertiary amine. In the human environments of the gastrointestinal tract, stomach, and bladder which generate the nitrosating agent, the derivation of various nitrosamines from these drugs has been presented. Dimethyl nitrosamine has been derived from methadone and diethylnitrosamine has been generated from chlorpromazine and chloroquine, respectively. Chlorpromazine, methadone, chloroquine, primaquine, and phenacetin have also produced by reaction mechanisms various nitrosamines of hitherto unknown carcinogenicity. The dimethylnitrosamine and diethylnitrosamine derived from methadone, chlorpromazine, and chloroquine are of proven carcinogenicity in experimental animals and they therefore constitute a hazard to humans.