Stability study of a new antidote drug combination (Atropine-HI-6-Prodiazepam) for treatment of organophosphate poisoning.

The main purpose of this study was to investigate the chemical stability of a new antidote combination for the treatment of organophosphate poisoning. The antidote combination was packed (enclosed) in two plastic compartments separated by a barrier film. One of them contained a powder oxime cholinesterase reactivator (HI-6-monohydrate 1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2-[(hydro xyimino)meth yl]-pyridinium dichloride). The other contained an anticholinergic (Atropine) and an anticonvulsant (Prodiazepam or Avizafone (L-lysyl-N-(2-benzoyl-4-chlorophenyl)-N-methyl-glycinamide dihydrochloride) drug in a liquid mixture. The plastic compartments were mounted in an autoinjector device to study the dissolution of HI-6 by ejection of the solution. Drug analysis was performed by high-performance liquid chromatography. The results obtained after 6 months show that this new antidote combination is stable. The amount of each antidote is unchanged during the study. Some known degradation products can be detected in small amounts. The autoinjector mechanism used, gives a complete dissolution of HI-6 powder in the liquid mixture throughout the study.

Micronuclei in lymphocyte subsets in relation to immune proteins and allergic disease.

This is an investigation of 54 boys and 23 girls with a median age of 19 years (range 18-22 years). The study group contained 12 boys and five girls with asthma and 23 boys and seven girls with allergic rhinitis. Sensitivity to pollen and furred animals were reported by 22 boys and eight girls and by 17 boys and six girls, respectively. The levels of serum immune proteins (IgA, IgE and IgG with subclasses, and IgM) were determined by immunological techniques. As a biomarker of chromosomal damages, the lymphocyte micronuclei was used. We analyzed the frequencies of micronuclei in 3000 B-lymphocytes and in equal numbers of T4- and T8-lymphocytes. The lymphocytes were separated by magnetic attraction in T4 (CD4), T8 (CD8) and B (CD19) fractions using Dynabeads(R). The most interesting finding of this investigation was that the three markers of atopic disease, asthma, hypersensitivity to pollen and IgE levels, associated significantly with increased frequencies of micronuclei in B-lymphocytes. There was also a relation between IgA and the frequency of micronuclei in B-cells. In an epidemiological study of 7000 individuals with allergic diseases, we have found an over-risk for lymphomas in the group with positive skin prick test. Hypothetically, we think that there may be a link between our present finding of an increased mutagenic activity and the lymphoma over-risk among individuals with allergic disease since most lymphomas stem from B-lymphocytes.

Increased frequencies of micronuclei in T8 lymphocytes of smokers.

Micronucleus frequencies and mitotic indices were analyzed in B, T4, and T8 lymphocytes from 40 smokers and 42 non-smoking referents. The highest level of micronuclei was found in T4 cells followed by T8 and B cells. These differences were statistically significant. There were statistically significant linear correlations between the micronucleus frequencies of all three subsets. There was a statistically significant effect of smoking only in the T8 cells. Smoking also increased the number of neutrophilic granulocytes and lymphocytes in the peripheral blood. There was a statistically significant effect of age on the micronucleus frequencies in T4 and T8 lymphocytes. The mitotic indices did not have any effect on the micronucleus frequencies and they were not influenced by smoking, age or sex.

Micronuclei and mitotic index in B-, T4- and T8-cells treated with mitomycin C and gamma-irradiation.

Lymphocytes were treated in vitro with mitomycin C and gamma-radiation at different doses (0-250 nmol/l and 0-2 Gy, respectively). After incubation in RPMI 1640 medium and stimulation with phytohemagglutinin for 72 h, the lymphocyte subgroups T4 (CD4), T8 (CD8) and B (CD19) were separated by an immunomagnetic method and analyzed for the presence of micronuclei. With mitomycin C the highest levels were found in T4- and B-cells. When micronuclei were induced by irradiation the T4-cells showed the highest frequencies and the B-cells the lowest. The outcome of B-cells with gamma-irradiation was probably due to a pronounced cytotoxic reaction in this cell type, which could be measured as a decrease in mitotic index.

Effect of acid precipitation on retention and excretion of elements in man.

From a population of 8918 farmers, 237 were selected whose consumption of locally produced foods was high. The subjects' water sources, private wells, were of different degrees of acidity. Significant associations between pH (median 6.7, range 4.7-8.6) of the drinking water and element concentrations were found. The correlation was negative for aluminium (Al; median 0.07 mumol/l), cadmium (Cd; 0.44 nmol/l), copper (Cu; 0.24 mumol/l) and lead (Pb; 1.9 nmol/l), and positive for calcium (Ca; 0.62 mmol/l) and magnesium (Mg; 0.21 mmol/l). Associations could not be found between the pH of, or element concentrations in, the water and concentrations of A1 (0.17 mumol/l), Mg (0.86 mmol/l) and selenium (Se; 1.0 mumol/l) in plasma, Cd (2.0 nmol/l), Pb (0.19 mumol/l) and mercury (Hg; 13 nmol/l) in blood, or A1 (12 mumol/mol creatinine) and Cu (11 mumol/mol creatinine) in urine. The concentrations of Hg in blood and Se in plasma were related to fish consumption, Cd and Pb in blood to smoking, A1 in urine to antacid intake, Pb in blood to rifle activities and hunting, and Hg in blood to hunting. Acid precipitation has an effect on element concentrations in drinking water, but not on the retention of those elements in the subjects investigated.

Increased frequency of micronuclei in lymphocytes of Swedish chimney sweeps.

Forty-five Swedish chimney sweeps and 49 controls were investigated with micronuclei stimulated by phytohemagglutinin and pokeweed mitogen and by analysis of lymphocyte subgroups and neutrophilic leukocytes. There was a statistically significant effect among sweeps with respect to both micronuclei variables and neutrophilic leukocytes. The effect on lymphocyte micronuclei was most prominent in pokeweed mitogen-stimulated cells, which may imply that the T4 lymphocytes were preferentially damaged by the occupational exposure.

B- and T-lymphocyte micronuclei in chimney sweeps with respect to genetic polymorphism for CYP1A1 and GST1 (class Mu).

Epidemiological studies have shown an increased incidence of lung cancer, bladder cancer, and esophageal cancer in chimney sweeps, probably due to their exposure to PAH in soot. The work environment for sweeps has, however, improved during the last decades. It was thus important to assess whether the present exposure still may cause genotoxic effects. A further objective was to assess whether genetic polymorphisms in metabolic enzyme activities could explain some of the variation in the parameters of genotoxicity. Venous blood samples were drawn from 71 chimney sweeps and 59 control subjects. Micronuclei were analyzed in activated peripheral B- and T-lymphocytes with preserved cytoplasm. Polymorphisms for CYP1A1 and GST1 in the sweeps were analyzed by a PCR technique. The sweeps did not have higher frequencies of micronuclei in B- or T-lymphocytes than the control subjects, when allowance was made for age and smoking in a multiple regression analysis. Further, there was no association between years of active work as a sweep and any of the two micronucleus parameters. None of the sweeps had the rare CYP1A1 genotype val/val and only one individual had the m2/m2 genotype. The presence of at least one GST1 allele (GST1+) was observed in 36 subjects (51.4%). Thirteen individuals (18.6%) were of the m1/m2 or m2/m2 genotype. And among those only seven had the combined GST1- and m1/m2 genotype. No difference was observed in B- or T-lymphocyte micronucleus frequencies between sweeps with the rare CYP1A1 genotypes m1/m2, m2/m2 or ile/val compared to individuals with the m1/m1 and ile/ile genotypes. Moreover, the GST1 deficient sweeps (GST1-) did not show any altered micronucleus frequency compared to the GST1 positive sweeps. A possible reason for the lack of genotoxic effect in sweeps is the improved hygienic conditions and change in fuels, which has decreased the exposure levels for PAH. Host polymorphisms for metabolizing enzymes did not influence the micronucleus frequencies. As the sweeps did not differ from the control subjects, with respect to micronucleus frequencies, no conclusion on the importance of host polymorphisms for genotoxic risk can be drawn.

Micronucleus induction in human B and T lymphocytes separated by an immunomagnetic technique.

Micronuclei were induced in human lymphocytes by mitomycin C or gamma radiation. After the incubation the cells were separated in B- and T-cells by an immunomagnetic method, i.e., Dynabeads. The cultures were stimulated by PHA. The effect of different incubation times (60-110 h) as well as different doses of mitomycin C (0-100 nmol/l) and gamma radiation (0-2 Gy) at an incubation time of 72 h were studied. There were significantly higher frequencies of micronuclei in B- than in T-cells, when micronuclei were induced by mitomycin C. This was not found with gamma radiation, probably because of a cytotoxic effect of this agent on the B-cells.

Cytogenetic effects in rotogravure printers exposed to toluene (and benzene).

Comparing 21 rotogravure printers exposed to toluene (medians: time-weighted air level 150 mg/m3, blood toluene 1.6 mumole/l) and 21 unexposed controls (median blood toluene less than or equal to 0.01 mumole/l) there was a significant increase in the frequency of micronuclei (MN) in pokeweed mitogen (PWM)-stimulated peripheral blood lymphocytes in the printers, as compared to the controls (2.8% vs. 1.5%; p = 0.03; all p adjusted for age and smoking). The frequency of small MN (size ratio MN/main nucleus less than or equal to 0.03) in PWM-stimulated lymphocytes was associated with the exposure (1% vs. 0.3%; p = 0.05). Furthermore, among the exposed subjects there was an association between blood toluene and small MN (0.17% per mumole/l; p = 0.0005). Small MN in phytohemagglutinin (PHA) cultures displayed no association with any exposure parameter. However, in the printers, an estimated cumulative exposure index was weakly correlated with the frequency of total MN in PHA-stimulated cells (0.00003% per mg/m3 x year; p = 0.07). Among the printers, chromosomal breaks in PHA-stimulated cells were associated with the duration of earlier benzene exposure (0.03% per year; p = 0.01). The results of this study strongly indicate that toluene causes a clastogenic effect on the B-cells even at low exposure levels. Further, earlier benzene exposure seems to have caused chromosomal breaks in T-cells.