Re-colorization of museum specimens: a modification of Romhanyi's technique based on pyridine/nicotine hemochromogen reactions.

Romhanyi's technique for preservation and restoration of color in museum specimens is almost unknown outside Hungary. The technique was based on the use of pyridine, nicotine and sodium dithionite (sodium hydrosulphite) for restoring the natural red color of formalin-fixed gross specimens. We developed a modification of the original technique using a mounting medium containing citrate-phosphate-dextrose (CPD) buffer and glycerin to control pH and osmolality. Specimens re-colorized with the modified and improved Romhanyi technique remained stable with a natural red color, and no histological deterioration, for more than 18 years. The technique demonstrates the formation of red colored complexes between nicotine and pyridine and ferro-hemochromes such as hemoglobin, myoglobin and other cytochromes in tissues, giving specimens a natural color. The modified technique enables further improvement for the old process of re-colorization of museum specimens. In addition, it opens up new methods for demonstration of nicotine, pyridine and their analogues in biological tissues.

The lessons of Bhopal [toxic] MIC gas disaster scope for expanding global biomonitoring and environmental specimen banking.

Bhopal Toxic gas tragedy represents one of the worst chemical accidents of the world. Autopsy and toxicological studies, apart from presenting evidence of acute and even chronic cyanide toxicity, provided a unique example of the incriminated chemical being traced to the bodies of the victims. The entry of methyl isocyanate (MIC) into the blood stream was established by the presence of carbamoylated end-terminal amino acids of haemoglobin and other tissue proteins. The presence of MIC trimer and a few other identified as well as unidentified tank residue constituents in the blood and viscera further established a close nexus of the products of pyrolysis of MIC in the aerosol inhaled by the victims. The Bhopal studies exemplify the scope for biological monitoring (BM) and environmental specimen banking (ESB) in chemical accidents as part of the global efforts.

Acute histopathological changes induced by methyl isocyanate in lungs, liver, kidneys & spleen of rats.

Methyl isocyanate (MIC), inhaled or administered subcutaneously (sc) at lethal concentration/dose caused essentially similar histopathological changes in all the viscera except for the lungs. The observed congestion of the viscera, foci of hepatocellular necrosis with widening of Disse's spaces in the liver and tubulo-rhexis with degeneration in the kidneys are attributable mostly to the initial shock. In addition, the lungs revealed more distinct route specific patterns of histopathological lesions. Inhaled MIC caused acute eosinophilic necrosis of the bronchial epithelium and frank alveolar edema, while MIC administered sc led to prominent vascular endothelial damage and severe interstitial pneumonitis with normal bronchial epithelium. The differential loci of damage in the lungs may be attributed to the immediate contact surface available for interaction with MIC.

Isolation of an unknown compound, from both blood of Bhopal aerosol disaster victims and residue of tank E-610 of Union Carbide India Limited--chemical characterization of the structure.

A total of more than 28 chemical entities/reaction products in the form of gases, vapour and particulate matter were reported from the tank E-610 of methyl isocyanate (MIC) storage tank of Union Carbide India Limited on the night of 2/3 December 1984 in Bhopal. In earlier studies, methyl isocyanate and its trimer, with a few other compounds, were reported in the human victims preserved in deep freeze. Randomly selected samples were analysed by gas chromatograph coupled with mass spectrometer (ITD-800, Finnigan MAT, UK). Four of the cases showed the peaks and fragmentation pattern identified with one of the unidentified compound of molecular weight 269 amu in the Tank Residue, which constituted about 0.2 area per cent on GC-ITD. After isolation by column chromatography and being exposed to characterization, it was identified as a Spiro compound. It was possibly formed by the polymerization of five molecules of methyl isocyanate.

Comparative toxicity of methyl isocyanate and its hydrolytic derivatives in rats. I. Pulmonary histopathology in the acute phase.

The present study describes the acute histopathological changes induced by methyl isocyanate (MIC) in the lungs of rats at 24 h after a single exposure to varied concentrations/doses of MIC by inhalation and subcutaneous (s.c.) routes and also delineates the effects due to the hydrolytic derivatives of MIC, viz., methylamine (MA) and N,N'-dimethyl urea (DMU). MIC, either inhaled or administered s.c., resulted in a wide range and extent of histopathological changes in the lungs, proportional to the exposure concentration/dose. The salient, effects of inhaled MIC are acute necrotizing bronchitis of the entire respiratory tract accompanied by varying degrees of confluent congestion, hyperemia and interstitial and intra-alveolar edema, while MIC administered s.c. led to prominent vascular endothelial damage, congestion and severe interstitial pneumonitis with apparently normal bronchial epithelium; and intra-alveolar edema only with the high dose. The only noteworthy lesion produced by MA and DMU (to some extent) was interstitial pneumonitis, suggesting their possible involvement in the subsequent inflammatory response of MIC. Except, for the endothelial changes, the overall spectrum of the histopathological lesions is quite comparable to those observed in the lungs of Bhopal victims during the acute phase.

Comparative toxicity of methyl isocyanate and its hydrolytic derivatives in rats. II. Pulmonary histopathology in the subacute and chronic phases.

This paper describes the long-term (subacute and chronic) histopathological effects in the lungs of rats subjected to a single exposure to methyl isocyanate (MIC) by both the inhalation and subcutaneous (s.c.) routes as well as the role of methylamine (MA) and N,N'-dimethyl-urea (DMU), the hydrolytic derivatives of MIC in eliciting the observed changes. At the subacute phase, the intraalveolar and interstitial edema were prominent only in the inhalation group as against the more pronounced inflammatory response in the s.c. route. With the progress of time the evolution of lesions appeared to be similar, culminating in the development of significant interstitial pneumonitis and fibrosis. MA, one of the hydrolytic derivatives of MIC, also caused interstitial pneumonitis progressing to fibrosis, albeit to a lesser extent than MIC, indicating its contribution to the long-term pulmonary damage. The diffuse interstitial pulmonary fibrosis observed at 10 weeks after a single exposure to MIC by either route is of greater significance in the context of the occurrence of pulmonary fibrosis in the late autopsies of Bhopal gas victims and also clinical sequelae in some of the survivors.

GC-NPD and GC-MS analysis of preserved tissue of Bhopal gas disaster: evidence of methyl carbamylation in post-mortem blood.

Twenty-five preserved autopsy blood samples of Bhopal toxic gas exposed victims were analysed by gas chromatography (GC) coupled with either Nitrogen-Phosphorous detector (NPD) or mass spectrometer (MS) for the presence of methyl carbamyl valine in terms of valine methyl hydantoin (VMH). 84% of these samples showed a positive test for VMH on GC-NPD and the identity of the peaks were further confirmed on GC-MS. The concentration of VMH in the gas-affected positive blood samples ranged from 2.56 to 51.28 nanomoles. These results indicate entry of methyl isocyanate (MIC), one of the constituents of the toxic cloud caused by the disaster, into the blood stream of victims who had inhaled gas.

GC-MS identification of MIC trimer: a constituent of tank residue in preserved autopsy blood of Bhopal gas victims.

Based on the external and internal findings of Bhopal gas disaster victims, it was apparent that the gases and particulate matter came out as an aerosol. This was possibly the pyrolysed, reformulated, reconjugated suspension of constituents of the tank E-610 of Union Carbide India Limited, Bhopal, while it was claimed to be methyl isocyanate (MIC) only. It was postulated by the manufacturer of MIC, that the material inhaled by the victims of the Bhopal gas disaster does not cross the lung barrier (UCC press conference on 14th December 1984). It was observed that the more the victims ran, the more aerosol they inhaled and the fatalities were observed in such victims. The tissues, which were preserved in the deep freeze, were randomly selected and analysed by GC coupled with MS (ITD) Finnigan MAT, UK. 14 out of 34 autopsy cases showed MIC trimer peak in extracts of blood. This was one of the constituents of the aerosol and was also located in the tank residue, thereby proving that the trimer had passed the lung barrier.

Animal transmission of enteric non-A, non-B hepatitis infection to Macaca mulatta by faeco-oral route.

M. mulatta monkeys were inoculated faeco-orally by enteric non-A, non-B virus to study the development of clinical, biochemical, histopathological and serological changes in the blood and liver. Pooled stool samples positive for putative non-A, non-B viral antigen by micro-ELISA and aggregated viral particles by immune electron microscopy, were administered in two M. mulatta monkeys. Biochemical, histopathological and serological changes were seen in the blood and liver and excretion of 27 nm virus like particles around 27 days of inoculation in the experimental monkey but not in the control animal.

Gas chromatographic studies of the carbamylation of haemoglobin by methyl isocyanate in rats and rabbits.

Carbamylation of the N-terminal valine of haemoglobin with methyl isocyanate in rats and rabbits has been demonstrated in vitro and in vivo by gas chromatography. N-Methylcarbamylated haemoglobin, converted by cyclization into 3-methyl-5-isopropylhydantoin, has been quantified by gas chromatography. Standard hydantoin was synthesized, chemically characterized and used for calibration. The method is simple and reliable in the concentration range 0.06-2 nmol. Carbamylation of haemoglobin by methyl isocyanate in vivo in rats can be identified only above a dose of 1.05 mg/l in inhalation exposures. It is inferred that methyl isocyanate in the "active" form crosses the alveolar and erythrocyte membranes and carbamylates the haemoglobin.