HLö 7 dimethanesulfonate, a potent bispyridinium-dioxime against anticholinesterases.

HLö 7 dimethanesulfonate (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2,4-bis [(hydroxyimino)methyl]pyridinium dimethanesulfonate) is a broad-spectrum reactivator against highly toxic organophosphorus compounds. The compound was synthesized by a new route with the carcinogenic bis(chloromethyl)ether being substituted by the non-mutagenic bis(methylsulfonoxymethyl)ether. The very soluble dimethanesulfonate of obidoxime was also prepared by this way. HLö 7 dimethanesulfonate is the first water-soluble salt of HLö 7 that should be suitable for the wet/dry autoinjector technology, because aqueous solutions of HLö 7 are not very stable (calculated shelf-life 0.2 years when stored at 8 degrees C, 1 M solution, pH 2.5). The crystalline preparation contains 96% of the syn/syn-isomer, less than 2% of the syn/anti-isomer and some minor identified by-products. HLö 7 was very efficient in reactivating acetylcholinesterase (AChE) blocked by organophosphates as long as ageing did not prevent dephosphylation. HLö 7 was superior to HI 6 (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2- [(hydroxyimino)methyl]pyridinium dichloride) in reactivating soman and sarin-inhibited AChE from erythrocytes, and literature data indicate that HLö 7 exceeds HI 6 by far in reactivating tabun-inhibited AChE. In atropine-protected, soman-poisoned mice HLö 7 was three times more potent than HI 6 (protective ratio 5 versus 2.5), and in sarin-poisoned mice HLö 7 was 10 times more potent than HI 6 (protective ratio 8 for both oximes). In atropine-protected guinea-pigs HLö 7 was less effective than HI 6 (protective ratio: 2.3 versus 5.2 for soman; 5.2 versus 6.8 for sarin; 4.3 versus 3.8 for tabun). The mean survival time of anaesthetized guinea-pigs exposed to 5 LD50 soman (6.3 min) was increased by atropine (27 min) and atropine + HLö (57 min). HLö 7 alone did not prolong the survival. The most impressive effect of HLö 7 was on respiration: 3 min after i.v. injection of HLö 7 and atropine, the depressed respiration increased rapidly to 60% of control and remained at that level during the observation period (60 min). With atropine alone, respiration recovered only slowly. Behavioural and physiologic parameters were determined in atropine-protected mice exposed to a sublethal soman dose. The running performance was significantly improved by HLö 7. Even central symptoms, e.g. hypothermia and convulsions, were decreased markedly by HLö 7 (evaluation 60 min after poisoning). The pharmacokinetic data for HLö 7 in male beagle dogs are similar to those of HI 6.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacokinetics and efficacies of obidoxime and atropine in paraoxon poisoning.

The efficacies (ED50) of obidoxime and atropine against paraoxon poisoning in mice were determined by administering the antidotes 5, 20, 40 and 60 min before administration of the organophosphate. With increasing time intervals t between the administration of the antidote and paraoxon, the dose of antidote (ED50), that reduced the lethality of 2 LD50 of paraoxon to 50% increased. The standardized log ED50/t plot was linear and yielded the "efficacy half-life". In addition, the blood concentrations c of the antidotes were measured, resulting in a linear log c/t plot. The "efficacy half-life" was approximately twice the half-life of the antidote in blood. The possible reasons for this phenomenon are discussed.

Carcinogenicity of sodium dichromate and chromium (VI/III) oxide aerosols inhaled by male Wistar rats.

In inhalation chambers, male Wistar rats of the strain TNO-W74 were continuously exposed to submicron aerosols of sodium dichromate and to a pyrolyzed Cr(VI)/Cr(III) (3:2) oxide mixture. The sodium dichromate (Na2Cr2O7) aerosol had the chromium concentrations of 25, 50 and 100 micrograms/m3, the chromium oxide mixture (Cr5O12) had the chromium concentration of 100 micrograms/m3. After 18 months of inhalation the rats were held under conventional conditions for a further year. The experimental groups consisted of 20 rats and the control group of 40 rats. More than 90% of the rats in each group reached 2 years. At the end of the study the mortality rates amounted to 35%, 45% and 25% in the 3 sodium dichromate aerosol groups, respectively, and 50% in the chromium oxide mixture aerosol group, which was not significantly different from that of the controls (42.5%), living under the same conditions in filtered fresh air. In all sodium dichromate exposed groups significant effects were neither found clinically nor from hematology and clinical chemistry compared to the controls. In the chromium oxide mixture group, however, there was a number of significant findings. Elevated white and red blood cell counts and serum cholesterol as well as decreased serum total immunoglobulin levels at different stages of the study were observed together with few local lung effects determined histopathologically in this group. We assume that these effects are mainly due to the increased chromium lung burden of the rats. At the end of the study the lung chromium retention was about 10 times higher for the rats exposed to chromium oxide versus sodium dichromate at an aerosol Cr-concentration of 100 micrograms/m3, while the kidney chromium retention was measured to be nearly equal in both groups. Three primary lung tumors (2 adenomas and 1 adenocarcinoma) and 1 malign tumor of the pharynx were found at the highest Cr-concentration (100 micrograms/m3) of the sodium dichromate aerosol, 1 primary adenoma of the lung was in the chromium oxide mixture group exposed also to a Cr-concentration of 100 micrograms/m3. No primary lung tumors were observed in the other experimental and control groups. These results indicate a weak carcinogenicity at 100 micrograms/m3 for the rats continuously exposed to submicron Na2Cr2O7 and Cr5O12 aerosols. Thus, there may be a small carcinogenic risk from occupational relevant chromium air levels. However, results have to be confirmed with larger animal populations.

The protective effect of oxime pretreatment upon the inhalative toxicity of sarin and soman in rats.

The functional set-up of a static inhalation device is described. The closed system consists of a reservoir for the circulating test gas, an inhalator for "nose-only" exposure of 10 rats, and analyzers for the test gas and CO2. It has an overall volume of 200 liters. The following LCt50 values for rats exposed to noxious vapors were determined: DFP 3629, sarin 191, and soman 211 mg X min X m-3. Pretreatment with atropine (10 mg/kg) together with the oximes Toxogonin or HJ6 (each 13 mg/kg) 10 min before the exposure raised the LCt50 of sarin by a factor of 2.5 and 14, respectively. In case of soman poisoning, only HJ6 (50 mg/kg) together with atropine was effective, which increased the LCt50 by a factor of 7.

[The effect of organophosphates on heart ATPase in the rat]. / Wirkung von Organophosphaten auf Herz-ATPasen von Ratten.

The effect of organophosphate intoxication with diisopropylfluorophosphate (DFP), paraoxon and parathion on the activity of some ATPases of rat hearts was examined. Biochemical and histochemical methods were employed to determine the activities of the transport ATPase, of the myofibres and mitochondrial ATPases using different doses and antidote application. The organophosphates studied here had an inhibitory effect on the activities of enzymes. Biochemical measurements showed that the ATPase activities are dependent upon the duration of poisoning, the dose of poison and the applied organophosphates. DFP caused the highest restriction in activity of the examined ATPases. The Ca2+-stimulated ATPase was more sensitive than the Na+/K+-ATPase to the application of organophosphates. The antidote therapy determined the grade of influence on activity. Combined treatments of organophosphate poisoning with atropine and obidoxime chloride in the high doses of 7.5 mg/kg and 12.5 mg/kg body weight i.m. proved to be the most effective against the organophosphate intoxication. Antidote combination of small doses and also single application of atropine or obidoxime chloride might cause a stimulation of activity of the Na+/K+-ATPase and a significant decline of the Ca2+-ATPase.

Carcinogenicity of cadmium chloride aerosols in W rats.

Lung cancers were induced in inbred W rats by cadmium chloride aerosols. For 18 months, 120 male W rats were continuously exposed to cadmium chloride aerosols with cadmium (Cd) concentrations of 12.5, 25, and 50 micrograms/m3, respectively. For the same period of time, 41 rats were kept in filtered air; these rats served as the control group. The survivors were killed 13 months after the end of the inhalation experiments. Histopathologic examination revealed a dose-dependent incidence of primary lung carcinomas of the following types: adenocarcinomas, epidermoid (squamous cell) carcinomas, combined epidermoid carcinomas and adenocarcinomas, and mucoepidermoid carcinomas. The incidence of lung carcinomas was 71.4% in the group exposed to 50 micrograms Cd/m3, 52.6% in the group exposed to 25 micrograms Cd/m3, and 15.4% in the group exposed to 12.5 micrograms Cd/m3. None of the controls developed lung carcinomas. At the end of the experiment, the remaining Cd concentrations in the lungs were relatively high, almost at the same level as those in the livers.

Effect of chronic alcohol consumption on tumor incidence due to dimethylnitrosamine administration.

To study the effect of chronic alcohol consumption on tumor development due to dimethylnitrosamine (DMN) administration, female Sprague-Dawley rats were pair-fed for 3 weeks a nutritionally adequate liquid diet containing either ethanol (36% of total calories) or isocalorically substituted carbohydrates as control diet. Thereafter, the animals were maintained on laboratory chow and tap water ad libitum for another 2 weeks and received 1.5 mg DMN i.p. per day for the first 5 days. This 5-week cycle was repeated three more times. Chronic treatment with an alcohol-containing diet was shown to significantly improve the mean survival time of DMN-treated rats compared with identically treated animals fed the control diet, but the total number of tumors observed under these experimental conditions and the target organ remained virtually unchanged.

Flow cytometric measurement of nuclear DNA content variations as a potential in vivo mutagenicity test.

Mutagen-induced variations of the cellular DNA content have been studied in mouse bone marrow cells in vivo using high resolution flow cytometric techniques. During the first days after a single injection of the chemical mutagen cyclophosphamide an increased coefficient of variation in the G1 peak of the flow histograms was observed. The magnitude and duration of this effect were dose-dependent. The reproducibility of the measurements was high, indicating that individual variability between animals and instrumental dispersion is small. The results demonstrate that on the basis of the flow cytometric measurement of cellular DNA content, a short-term in vivo test for mutagenicity can be established which is much faster than conventional cytogenetic methods.

[Disintegration of human spermatozoa in flow cytometry of semen]. / Desintegration von Spermatozoen bei der impulscytophotometrischen Analyse des menschlichen Spermas.

In some cases, the pretreatment of human spermatozoa produced desintegration of various degrees, while in others a reduced staining was observed. For the evaluation of these phenomena the following techniques were applied: papain/DTE solution at pH 5.5 and 6.4, pepsin solution and a staining procedure without any preceeding decondensation of the spermatozoa nuclei. Pathological spermatozoa from patients with severe disorders of spermatogenesis showed an extended resistance to the pretreatment procedures, the spermatozoa being stained after papain/DTE pretreatment at pH 5.5 only. Contrary to this finding, normal spermatozoa and spermatozoa with slight morphological aberrations desintegrated when papain/DTE solutions were used. In this group the abortion rate was high.

Flow cytogenetic studies in chromosomes and whole cells for the detection of clastogenic effects.

Flow cytometric measurements of the chromosomal DNA content have been used to develop a screening method for the detection of chemically- or physically-induced cytogenetic damage. The reproducibility of this flow cytogenetic assay was shown in a series of subcultures of a Chinese hamster cell clone. The accuracy and sensitivity was tested in cultures treated with chemical mutagens and x-rays. The clastogenic effectiveness was quantified and the dose-effect relationship was established by the increase of the coefficient of variation of the peak of the largest chromosome type in the flow histograms. Since structural chromosome aberrations cause an unequal division of the DNA at mitosis, it is expected that clastogenic effects can be detected also in whole cells of growing populations as an increased dispersion of the cellular DNA content. In order to test this feature, high resolution flow cytometric measurements were performed in x-irradiated hamster cells in vitro and mouse bone marrow cells in vivo.

Lung deposition and clearance of cadmium in rats exposed by inhalation or by intratracheal instillation.

115mCdCl2 was nebulized by a jet nebulizer, yielding an aerosol concentration of Cd of 660 ng/l. Rats were exposed nose only for 1 h. For the instillation study 115mCdCl2 was instilled intracheally, 8 microgram in 0.3 ml NaCl. Rats of both studies were serially sacrificed from day 0-100, and the lung activity was counted. The results showed a bi-exponential clearance pattern in both studies, where the long clearance gave half lives of 61 d (inhalation) and 66 d (instillation), respectively. The short term clearance half lives were 1.1 d (inhalation) and 0.7 d (instillation). About half of the deposited Cd is cleared in both studies in the fast phase. After inhalation, 16% more Cd was deposited in the alveolar area as compared to instillation. It was found that on the second day after instillation of CdCl2 only 2% could be lavaged out of the lungs, suggesting a protein binding of Cd in the lung.

Flow cytogenetics of uncloned and cloned Chinese hamster cells.

Flow cytometry has greatly facilitated the routine use of DNA content as a cellular indicator of the stages of the cell cycle and ploidy. DNA content can also be used to distinguish individual chromosomes. Fluorescent staining of chromosome DNA was done with a combination of ethidium bromide and mithramycin in hypotonic solution. Subsequent detergent treatment of the cells with Triton X-100 facilitated chromosome isolation. DNA flow cytometry of chromosomes of four established uncloned Chinese master cell lines showed 10 to 12 major subpopulations of chromosomes with varying degrees of overlap in the range of low and intermediate DNA content. Cloning of B14F28 cells, the line with the largest heterogeneity in chromosome number and DNA content, considerably reduced the dispersion in chromosome number and improved the resolution of DNA content distributions. Thus, cloned cells with a relatively homogeneous karyotype permit better discrimination of chromosome subpopulations by DNA content than uncloned cells and provide a more sensitive system to study mutagenic effects.

Decreased hepatotoxicity of dimethylnitrosamine (DMN) following chronic alcohol consumption.

Compared to controls receiving physiological saline, the i.p. administration of dimethylnitrosamine (DMN) on 5 consecutive days to rats fed a nutritionally adequate liquid diet resulted 24 hours after the last injection of significant increases in glutamic dehydrogenase (GDH), glutamic oxylacetate transaminase (GOT), and glutamic pyruvate transaminase (GPT) activities in the serum, indicating a striking hepatotoxic effect of this compound. This was confirmed by the histological demonstration of massive centrolobular necrosis. Conversely, following pretreatment of the rats with an ethanol containing liquid diet for 23 days and subsequent administration of DMN the increases of serum enzyme activities and massive centrolobular necrosis could not be observed. These results therefore suggest that chronic alcohol consumption protects from hepatotoxicity due to DMN, most probably due to an enhancement of detoxifying pathways of the parent component or one of its toxic metabolites.

Cellular radiation effects and hyperthermia: cytokinetic investigations with stationary phase yeast cells.

Wild type diploid yeast, Saccharomyces cerevisiae strain 211, was subjected to 250 kV X-rays or 50 degrees C heat treatment for 30 min or to a combination of both. X-ray exposure took place either in air or in nitrogen. Cell number, percentage of budding cells and cell cycle progression was followed for up to 12 h post irradiation. The distribution of cell cycle stages was determined by flow cytofluorometry. All treatments cause a retardation of cell division rate. Hyperthermia leads mainly to a lengthening of G1, whereas X-rays arrest the cells reversibly in G2. The effect of the combined treatment appears to be merely additive. No. selective action of hyperthermia on hypoxic cells was found.

[Analysis of spermatozoa with flow cytophotometric determination of DNA]. / Die Analyse von Spermaproben mit Hilfe impulscytophotometrischer DNS-bestimmungen.

Semen samples of fertility patients and fertile men were analysed by an improved technique of flow cytophotometry. The DNA-contents of mature spermatozoa were measured with a high level of accuracy. Besides spermatozoa, peaks of spermiogenetic cells and other seminal cells could be demonstrated. Complementary to the results of semen analyses the complex histograms of pulse cytophotometry investigations may give further informations on the nature of the underlying disturbances of spermatogenesis.

[Possible combinations of antidotal therapy in rats following intoxication with phosphoric acid esters (author's transl)]. / Möglichkeiten einer kombinierten Antidottherapie an Ratten bei Vergiftungen mit Phosphorsäureestern.

In order to improve or supplement the currently used therapy of poisoning with phosphoric acid esters, a protein free extract from calf blood containing low-molecular subtances (Actihaemyl, Solcseryl) has been tested in female Wistar rats. After poisoning with paraoxon, DFP and soman, a combination of Solcoseryl, Toxogonin and atropine sulfate prolonged the time until exitus, improved the overall rate of survival and considerably shortened the phase of regeneration of the experimental animals even when they had been intoxicated with high doses of poison.