Amin A. Nomeir

Gossypol: High-performance liquid chromatographic analysis and stability in various solvents

An improved high performance liquid chromatography (HPLC) method of gossypol analysis was developed and compared with the current spectrophotometric method. Gossypol was determined in four samples of cottonseeds ofGossypium hirsutum with varying contents of gossypol, as well as in the stems and roots ofG. arboreum andG. thurber. The roots of both species each contained equally high amounts of gossypol, while the stems of each species varied in content. Those ofG. thurber contained slightly higher amounts of gossypol than those ofG. arboreum. Among the seeds studied, the glanded cottonseeds contained the highest concentration of gossypol, followed by low gossypol seeds, then glandless cottonseeds, and finally the roasted glandless cottonseeds. The stability of gossypol was studied in five solvents at six different storage temperatures. Both the type of solvent and the temperature were found to affect the rate of decomposition of gossypol. In all the solvents studied, gossypol was found to be highly unstable at 37 C and at room temperature, while its stability increased as the storage temperature decreased. At all temperatures, depending on the solvent used, the rate of decomposition increased in the following order: acetone < acetonitrile < chloroform < ethanol < methanol. Although the decomposition products of gossypol were not identified, their formation was found to be dependent on the solvent used.

The role of pharmacokinetics and metabolism in species sensitivity to neurotoxic agents

Attempts have been made to review the role of pharmacokinetics and metabolism in species and age sensitivity as well as the development of various toxic conditions of some neurotoxic chemicals. The route of administration may play a prominent role in the development of various toxic effects of some organophosphorus compounds such as DEF. Such variation was attributed to the differential metabolism which was found to be highly dependent on the route of administration. It is obvious from the data presented here that animals that are sensitive to OPIDN are less active in the metabolism and elimination of the neurotoxic chemical and/or its metabolite(s). So, a compound may stay for a longer period in the body of the sensitive animals resulting in greater accessibility of target tissues to the deleterious effects of the neurotoxic compounds. However, many of these neurotoxic chemicals require metabolic activation to exert their effect. While the insensitive species may convert the compound to its active metabolite faster than that of the insensitive species, this is circumvented by the far greater capability of the insensitive animals to metabolize the active metabolite and/or the parent compound to less toxic, more polar, excretable metabolites. However, it must be stressed that these studies are far from complete, and caution should be exercised in interpreting and correlating many of these results. It is difficult, and sometimes misleading to compare data from various studies due to differences in dosage, the number of animals used, route of administration, experimental protocols, etc. With respect to hexacarbons, species sensitivity is obvious, but not as extensively investigated as OPIDN. To our knowledge, no studies are available addressing species difference in pharmacokinetics and metabolism of these chemicals. The data presented in this review suggest that metabolism and pharmacokinetics may play an important role in the development of OPIDN. However, this does not rule out the influence of other factors such as target sensitivity. This necessitates further qualitative and quantitative metabolic studies which are carefully planned to address these issues.

Comparative toxicity, cholinergic effects, and tissue levels of S,S,S,-tri-n-butyl phosphorotrithioate (DEF) to channel catfish (ictalurus punctatus) and blue crabs (callinectes sapidus)

Abstract The acute neurotoxic effects of S,S,S-tri-n-butyl phosphorotrithioate (DEF) on juvenile channel catfish and adult blue crabs was examined using short-term exposures in aerated, static aquaria. The effects on acetylcholinesterase (AChE) activity in catfish brain and skeletal muscle, and in crab ganglia, was studied. In addition, recovery of AChE activity was followed after transferring the animals to DEF-free water. The results indicate that both species exhibit pronounced anticholinesterase effects due to DEF. Recovery of AChE activity is quite slow in nervous tissues of both animals and catfish skeletal muscle, with effects persisting for several weeks following a single acute exposure. Tissue levels of DEF, as well as its rate of disappearance, were followed in several tissues of each species. The highest levels were detected in fish liver, but high levels were also detected in fish brain. Levels in crab ganglia were an order of magnitude lower than those in fish brain, but the anticholinergic effects were similar in both species. Disappearance was biphasic from tissues in both animals, with relatively long half-lives in target tissues.

Studies on the metabolism of the neurotoxic tri-o-cresyl phosphate. Distribution, excretion, and metabolism in male cats after a single, dermal application

The metabolism of a single, dermal dose of 50 mg/kg of [14C]tri-o-cresyl phosphate (TOCP) was studied in male cats. TOCP was applied to an unprotected, preclipped area on the back of the neck. Three animals were sacrificed on each of 0.5, 1, 2, 5, and 10 days following application. Radioactivity disappeared biexponentially from the dosing site with a faster initial rate; 73% of the dose disappeared in the first 12 h followed by a slower phase with a half-life of 2.03 days. No radioactivity was detected in the expired air. TOCP was absorbed from the skin and subsequently distributed throughout the body. Generally, the highest concentrations of radioactivity were associated with bile, gall bladder, urinary bladder, kidneys, and liver; the lowest were found in the neural tissues, muscle, and spleen. Within the 10-day experimental period, approximately 28% and 20% of the applied dose were recovered in the urine and feces, respectively. TOCP and its metabolites in the urine, feces, bile, and plasma were analyzed by high performance liquid chromatography and liquid scintillation counting. TOCP was the predominant compound in the feces (26.3% of total fecal radioactivity); it was detected in a smaller percentage in the urine (2.3% of total urinary radioactivity). The major metabolite in the urine was o-cresol followed by di-o-cresyl hydrogen phosphate and o-cresyl dihydrogen phosphate; in the feces di-o-cresyl hydrogen phosphate was the predominant metabolite followed by o-cresyl dihydrogen phosphate. Trace amounts of saligenin cyclic-o-tolyl phosphate, hydroxymethyl, and di(hydroxymethyl) TOCP were also detected in the urine and feces. Other metabolites identified in the urine and feces were the stepwise oxidation products of the methyl group of o-cresol. Unlike the feces, the bile contained mostly metabolites with only trace amounts of TOCP detected at 12 h and 24 h following application. o-Cresyl dihydrogen phosphate and di-o-cresyl hydrogen phosphate were the prevalent metabolites in the bile and plasma. While di(hydroxymethyl) TOCP was present in trace amounts in plasma, an appreciable amount of saligenin cyclic-o-tolyl phosphate, believed to be the active neurotoxic metabolite, was detected. This study shows that skin is an important port of entry for TOCP. Since TOCP represents organophosphorous chemicals capable of producing delayed neurotoxicity in test animals and in humans, it is essential that industrial hygiene control prevents skin contamination of workers handling these chemicals.

Studies on the metabolism of the neurotoxic tri-o-cresyl phosphate. Synthesis and identification by infrared, proton nuclear magnetic resonance and mass spectrometry of five of its metabolites☆

Five metabolites of the industrial neurotoxic chemical tri-o-cresyl phosphate (TOCP) were synthesized and their structures were verified by infrared, IR; proton nuclear magnetic resonance, 1H-NMR; and mass spectrometry. The 2 acids, o-cresyl dihydrogen phosphate and di-o-cresyl hydrogen phosphate were prepared in 2 steps. Step 1, POCl3 was reacted with o-cresol, using 1:1 and 1:2 molar ratios, in the presence of anhydrous AlCl3 as a catalyst, to form the 2 intermediates o-cresyl phosphorodichloridate and di-o-cresyl phosphorochloridate, respectively. Step 2, the chloridate intermediates were hydrolyzed under the appropriate condition to the corresponding acids. These acids were further derivatized to the corresponding methyl ester and the products were analyzed by the spectroscopic techniques. Saligenin cyclic-o-tolyl phosphate [2-(o-cresyl)-4H-1:3:2-benzodioxaphosphoran-2-one] was synthesized by reacting the potassium salt of o-hydroxybenzyl alcohol with o-cresyl phosphorodichloridate. Hydroxymethyl TOCP [di-o-cresyl o-hydroxymethylphenyl phosphate] and dihydroxymethyl TOCP [o-cresyl di-o-hydroxymethylphenyl phosphate] were synthesized by reacting di-o-cresyl phosphorochloridate with the potassium salt of o-hydroxybenzyl alcohol. The products were separated and purified by repeated preparative thin-layer chromatography (TLC) using 3 different solvent systems. The purity of the 5 metabolites, which was determined by high performance liquid chromatography (HPLC), ranged from 92% to 99%.

Photodecomposition of Gossypol by Ultraviolet Irradiation

Decomposition of gossypol as a thin film or as a solution by ultraviolet irradiation was studied. The decomposition of gossypol followed monophasic exponential kinetics in which the rate of decomposition varied and depended upon the irradiation condition. The lowest rate of gossypol decomposition was observed as a thin film which showed a half-life of 97 min, while the highest rate was attained as a solution in acetone as indicated by a half-life of 4.5 min. Solutions in methanol and ethanol showed relatively lower rates of decomposition with similar half-lives of approximately 50 min. Acetonitrile and chloroform solutions showed intermediate rates of decomposition for gossypol with half-lives of 15 and 19 min, respectively. Although the degradation products of gossypol were not identified, their HPLC profiles were characteristic of the solvent used. HPLC profiles of gossypol degradation products in methanol, ethanol, acetone and acetonitrile were similar, each exhibiting two peaks with variable ratios depending on the solvent and the time of exposure. The degradation products of gossypol when irradiated as a thin film and as a solution in chloroform were different from those in other solvents. In all cases, when gossypol and/or its degradation products were continuously exposed to ultraviolet radiation, they decomposed to products no longer having an aromatic structure.

Absorption, distribution, and elimination of a single oral dose of [14C]tri-o-cresyl phosphate in hens.

The absorption, distribution, elimination, and metabolism of a single oral dose of 50 mg (4.6 microCi)/kg of uniformly phenyl-labeled [14C]tri-o-cresyl phosphate (TOCP) was investigated in adult chickens. Three treated hens were killed at each time interval: 0.5, 1, 2, and 5 days. TOCP was absorbed from the gastrointestinal tract and subsequently distributed throughout the body. Generally, the highest concentrations of radioactivity were associated with gastrointestinal tract parts, bile, kidneys, liver, and lungs. Most of the radioactivity (47%) was excreted in the combined fecal-urinary excreta during the first 12 h. Very small fractions of the dose were deposited in egg albumen and egg yolk, 0.12% and 0.24%, respectively during the 5-day study. After 5 days, 99% of the dose was eliminated in excreta. TOCP and its metabolites in bile and the combined fecal-urinary excreta were analyzed by high-performance liquid chromatography and liquid scintillation spectrometry. TOCP and nine of its metabolites were identified. In the bile a TOCP active metabolite, saligenin cyclic-o-cresyl phosphate, was the predominant compound found compared to the parent compound in the excreta. These results suggest that in the hen TOCP is excreted slower than the rat and also undergoes metabolic activation. The absorption, elimination, and metabolic profile of TOCP in the hen may contribute to its sensitivity to delayed neurotoxicity.

Absorption, distribution, excretion and metabolism of a single oral dose of [14C]tri-o-cresyl phosphate (TOCP) in the male rat

A single oral dose of 50 mg/kg of [14C]TOCP was administered in corn oil to male rats. Three animals were sacrificed at each of 2, 6 and 12 h and 1, 2 and 5 days following dosing, and tissues and excreta were analyzed for 14C. Within 5 days, 63 and 36% of the dose were recovered in the urine and feces, respectively. Initially, the highest concentrations of radioactivity were observed in the gastrointestinal tract, its contents, the urinary bladder, liver and kidneys. Appreciable concentrations of 14C were detected in plasma, red blood cells, lungs and adipose tissues, while neural tissues, muscle, spleen and testes contained lower concentrations of radioactivity. Among neural tissues, the sciatic nerve contained the highest concentrations of 14C at all time points studied. The concentration of TOCP in plasma was at maximum by 6 h then declined biexponentially with terminal half-life of 46 h. The predominant metabolites in plasma were o-cresyl dihydrogen phosphate, di-o-cresyl hydrogen phosphate and o-hydroxybenzoic acid (salicylic acid). Small concentrations of the neurotoxic metabolite of saligenin cyclic-o-tolyl phosphate, were detected in plasma at all but the last time point analyzed. Most of the radioactivity extracted from the livers of rats sacrificed at 2 and 4 h were metabolites. No TOCP was detected in the urine or feces collected within 3 days after dosing. The major metabolite in the urine and feces was o-cresyl dihydrogen phosphate followed by di-o-cresyl hydrogen phosphate, salicylic acid, o-hydroxybenzyl alcohol and o-cresol. This study supports the hypothesis that the insensitivity of the rat to TOCP-induced delayed neurotoxicity may be attributed, in part, to the disposition and metabolism of this chemical.

High-performance liquid chromatographic analysis on radial compression column of the neurotoxic tri-o-cresyl phosphate and metabolites.

A method utilizing high-performance liquid chromatography (HPLC) has been developed for the analysis of tri-o-cresyl phosphate (TOCP) and its possible metabolites, o-cresyl dihydrogen phosphate, di-o-cresyl hydrogen phosphate, o-hydroxybenzyl alcohol, o-cresol, saligenin cyclic-o-tolyl phosphate [2-(o-cresyl)-4H-1:3:2:benzodioxaphosphoran-2-one], salicylic acid, salicylaldehyde, hydroxymethyl TOCP (di-o-cresyl o-hydroxymethylphenyl phosphate), and dihydroxymethyl TOCP (o-cresyl di-o-hydroxymethylphenyl phosphate). TOCP and its possible metabolites were analyzed on a reverse-phase C18 cartridge fitted into RCM-100 radial-compression separation system. Compounds were separated using a linear gradient of 25 to 80% acetonitrile in 2% aqueous acetic acid at a flow rate of 1.3 ml/min in a period of 22 min. Quantification was achieved by monitoring the ultraviolet absorbance of the column eluates at 254 nm and measuring peak areas. Retention times and peak areas were highly reproducible for all compounds analyzed. The relationship between peak area and amount injected was linear over a 100-fold range for all compounds analyzed. The minimum detectable level was 3 ng for salicylaldehyde, 25 ng for o-hydroxybenzyl alcohol and salicylic acid, and 50 ng for the remaining compounds. A mixture of TOCP and its possible metabolites was added to samples of cat liver, kidney, and plasma and then extracted and analyzed. High recovery and reproducibility for most compounds was observed in tissues analyzed.

Analysis of n-hexane, 2-hexanone, 2,5-hexanedione, and related chemicals by capillary gas chromatography and high-performance liquid chromatography.

Analytical methods, using capillary gas chromatography and normal-phase high-performance liquid chromatography, were developed for the analysis of the neurotoxic chemicals n-hexane, 2-hexanone, and 2,5-hexanedione and their suspected metabolites. Two gas chromatographic methods, using a 50-m glass capillary OV 101 column and cyclohexane as an internal standard, were employed. In both methods, the injector and detector temperatures were 220 and 280 degrees C, respectively. In method I the following temperature program was used: isothermic at 50 degrees C for 30 min, followed by a temperature increase of 10 degrees C/min to a final temperature of 180 degrees C, which was then maintained for 7 min. This method was used to analyze the following compounds: n-hexane, 2,5-dimethylfuran, 2-hexanone, 3-hexanone, hexanal, 1-hexanol, 2-hexanol, 3-hexanol, 5-hydroxy-2-hexanone, gamma-valerolactone, 2,5-hexanedione, and 2,5-hexanediol. Method II, which was developed for n-hexane and eight of its more common metabolites, used the following temperature program: isothermic at 70 degrees C for 15 min, followed by a temperature increase of 40 degrees C/min to a final temperature of 220 degrees C, which was maintained for 5 min. A linear relationship between peak area and amount injected was observed over a 100-fold range. The minimum detectable amounts ranged from 0.05 to 1 microgram, depending on the compound. Normal-phase HPLC, using a 5-micron silica cartridge fitted into an RCM-100 radial-compression separation system, was utilized to analyze 2-hexanone and its metabolites 2,5-dimethylfuran, gamma-valerolactone, 5-hydroxy-2-hexanone, and 2,5-hexanedione.(ABSTRACT TRUNCATED AT 250 WORDS)