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This study was undertaken to compare the tumour promoting effects induced by 3,4,5,3',4'-pentachlorobiphenyl (PCB 126) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition, interactive effects in rats treated with combinations of PCB 126 and TCDD were studied. Partially hepatectomized female Sprague-Dawley rats were initiated with nitrosodiethylamin. After 5 weeks of recovery the promotion treatment started and continued for 20 weeks. The results from the present study demonstrate that PCB 126 elicit approximately 10% of TCDD's tumour promoting activity measured as enhancement of the development of gamma-glutamyl-transpeptidase-positive altered hepatic foci in the liver. The factor required for the PCB to match the response of TCDD was adopted as a toxic equivalency factor and was in this case 0.1, which is the same as the factor suggested by Ahlborg et al. (1994). In the groups treated with a mixture of PCB 126 and TCDD the tumour promoting effect indicated an additive response. This result suggests that PCB 126 and TCDD act by the same mechanistical pathway, which in turn, supports that the toxic equivalency factor-concept can be used for TCDD-like tumour promoters.

To study the effectiveness of a group intervention in a clinical setting designed to increase knowledge of disability and improve sibling relationship among siblings of children with disabilities.A self-selected sample of 54 younger and older siblings with typical development (ages 8-12 years) of children with attention deficit hyperactivity disorder (ADHD) (9), Asperger syndrome (7), autistic disorder (13), physical disability (8) and intellectual disability (17) participated in collateral sibling groups. The Sibling Knowledge Interview (SKI) and Sibling Relationship Questionnaire (SRQ) were administered pre- and post-intervention.SKI scores increased (p 0.001) from pre- to post-intervention when merged diagnostic groups were compared. Comparisons of SRQ pre- and post-intervention scores across diagnostic sibling groups showed significantly different (p 0.05) score patterns.The results were encouraging and contribute to further development of interventions meeting the needs of siblings of children with disabilities. In view of the limited empirical research on group interventions for siblings of children with disabilities future work is needed to investigate the effectiveness of such interventions. Particular attention should be given to siblings of children with autism and siblings of children with intellectual disability.

This study was done to generate kinetic data on individual congeners of chlorinated biphenyls in the low dose range, which could be of value in the risk assessment procedure. Male Sprague-Dawley rats were given a single oral dose of a mixture of polychlorinated biphenyls (CBs) containing either CBs 105, 118, 138, 153, 156, 157, 170, and 180 (A-mix) or CBs 28, 52, 77, 87, and 101 (B-mix). Liver, serum, and adipose tissue were collected after 6 h up to 135 days, from rats given the A-mix, and after 6 h up to 4 days from rats given the B-mix. CB concentrations were measured in liver, serum, and adipose tissue. In addition, this study provides kinetic data of one of the major CB metabolites, 4-hydroxy-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107). The low doses used resulted in serum CB concentrations similar to human background serum concentrations. In the A-mix experiment all CBs show high initial liver and serum concentrations followed by redistribution into adipose tissue. Differences between congeners were correlated to molecular weight. High molecular weight correlated to lower uptake and slower redistribution. During dynamic steady-state the tissue concentrations decreased with a calculated first order rate between 54-129 days for halving the concentrations (half-life). Most of the decrease in concentration was explained by the growth-related increase of tissue masses in general and adipose tissue in particular. In the B-mix experiment, the concentrations of CBs in adipose tissue decreased with between 25 and 59% from day 1 to day 4. These results show that the B-mix congeners, given at low dose, have longer half-lives than previously reported in high dose studies. Partition coefficients between body compartments are reported and for the first time a high and congener specific liver-to-serum ratio of CB 77 is observed.

The relative tumour promoting activity of three structurally and toxicologically diverse polychlorinated biphenyls (3,4,5,3',4'-penta- 2,3,4,3',4'-penta- and 2,4,5,2',4',5'-hexachlorobiphenyl) was measured in an initiation/promotion assay in nitrosodiethylamine-initiated female Sprague-Dawley rats. The congeners under study were administered by once-weekly subcutaneous injections for 20 weeks. Evaluation of the development of gamma-glutamyl transpeptidase (GGT)- and glutation transferase P (GST-P)-positive hepatic foci showed that all congeners promoted altered hepatic foci, although 3,4,5,3',4'-pentachlorobiphenyl was far more potent. The volume fraction of the liver occupied by GGT-positive tissue in the 3,4,5,3',4'-pentachlorobiphenyl-treated animals (100 micrograms/kg per week) was 23%, while the volume fractions of altered liver tissue in the rats treated with 2,3,4,3',4'-pentachlorobiphenyl (5000 micrograms/kg per week) and 2,4,5,2',4',5'-hexaCB (20,000 micrograms/kg per week) were 1.2 and 2.3, respectively. The enhancement of GGT- and GST-P-positive foci was accompanied by an increased incidence of histological changes in the livers.

Intoxications with organophosphorus compounds are normally treated with a mixture of atropine and an enzyme regenerating oxime. The addition of diazepam to the conventional drug therapy is reported to greatly improve the antidotal effect. The implication of the cholinergic system in such intoxications prompted us to study the effect of different combinations of antidotes on the acetylcholine (ACh) synthesizing system in mouse brain in vivo. The antidotes studied in this paper are diazepam, HI-6 and 1–hyoscyamine, the active enantiomer of atropine. Diazepam decreases the synthesis rate of ACh both when administered separately and in combination with 1–hyoscyamine and HI-6. This is in contrast to 1–hyoscyamine which, in addition to blocking muscarinic receptors, also increases the release and rate of synthesis of ACh, which probably is an unfavourable effect of the antidote. This might at least partly explain the advantage of combining 1–hyoscyamine and an oxime with diazepam in intoxications with anticholinesterases. Mice administered soman (0.75 x LD50), after pretreatment with the three-drug combination of antidotes, show no cholinergic symptoms despite a 50% increase in endogenous ACh. The rate of synthesis of ACh in these mice is in the same range as in animals administered diazepam alone. Mice administered the same dose of soman with no antidotal pretreatment suffer from severe tremor and salivation, and have a strongly reduced synthesis rate of ACh.

The polychlorinated dibenzo-p-dioxins/dibenzofurans 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) were studied for liver tumour promoting activity in a medium-term altered foci assay in nitrosamine-initiated female Sprague-Dawley rats. The congeners under study were administered by weekly subcutaneous injections at three dose levels for 20 weeks. Evaluation of gamma-glutamyltranspeptidase (GGT+), altered hepatic foci development, showed that all congeners studied acted as potent promoters of hepatocarcinogenesis. TCDD and PeCDD were virtually equipotent as enhancers of foci development while PeCDF displayed approximately ten per cent of the activity of the dioxins. Analysis of the dioxin- and furan-congeners by gas chromatography/mass spectroscopy (GC/MS) technique showed that the retention of PeCDD and PeCDF in liver tissue was approximately 7 and 20 times, respectively, as high as the retention of TCDD. Based on the concentration of the respective congener in liver tissue, PeCDD and PeCDF were 0.14 and 0.007 times as active as TCDD as promoters of foci development. The dose related enhancement of GGT+ foci development induced by the PCDD/PCDF congeners was accompanied by an increased incidence of histological changes in the liver.

Within the amount of space available, it is not possible to deal with all cholinomimetic agents. We have therefore limited ourselves to two compounds, THA (tetrahydroaminoacridine) and metrifonate. Due to its use in AD, a considerable number of clinical papers exist concerning THA, but basic toxicology data seem to be largely missing. With regard to metrifonate, a considerable number of both clinical and toxicological data exist due to its use as an insecticide and in the therapy of schistosomiasis. A short review of this compound was given at the last symposium on AD (Giacobini E and Becker R, 1988) and metrifonate was suggested as a possible treatment in AD. So far, only one publication has appeared in this particular field (Becker et al., 1990). This review will focus mainly on toxicological data in review papers. It also includes a section on butonate that can possibly be of use to achieve a prolonged cholinesterase inhibition (ChEI).

In intoxications by organophosphates conventional prophylaxis and therapy by atropine and oximes have shown to benefit from the addition of diazepam treatment. The implication of the cholinergic system in such intoxications prompted us to study the direct effects of diazepam on this system. In this context acetylcholine (ACh) turnover in mouse brain in vivo is a suitable cholinergic model. Turnover of ACh was studied by following the incorporation of Ch into ACh after i.v. injection of deuterated Ch. To study the functional muscarinic receptor pool, we have developed a method utilizing the pharmacologically 'active' antipode of atropine, l-hyoscyamine. By injecting this compound and measuring its concentration in the brain of mice, it is possible to study specific receptor binding. The concentration of l-hyoscyamine after equilibration is assumed to correspond to the size of the functional muscarinic receptor pool. At this concentration, l-hyoscyamine prevented oxotremorine (OT)-induced tremor, confirming its physiological relevance. The effects of diazepam on the ACh dynamics are consistent with diazepam's known potentiation of gamma-aminobutyric acid's inhibitory function in nerve transmission, with a decreased turnover rate of ACh and increased levels of ACh and Ch as results. The modulating effect of diazepam on the binding properties of muscarinic receptors is probably one of the mechanisms responsible for its profound effects in treatment of intoxications with anticholinesterases. Keywords: Antidotes, Cholinesterase inhibitors.

The organophosphorus compound 0,0-dimethyl-(1-hydroxy-2,2,2-trichloroethyl)-phosphonate was introduced as an insecticide, trichlorfon, in 1952 (Lorenz et al., 1955) and as a drug, metrifonate, in the treatment of schistosomiasis in 1960 (Lebrun and Cerf, 1960). This organophosphorus compound is unique in that it has been claimed not to be a direct acting cholinesterase inhibitor but being transformed nonenzymatically into an active component dichlorvos, 2, 2-dichlorovinyl dimethyl phosphate (DDVP). The evidence for this transformation has mostly been indirect. Recently it has been proved chemically and quantitatively that this transformation occurs in the animal body (Nordgren et al., 1978). Metrifonate is the sole organophosphorus compound currently studied clinically in schistosomiasis. A substantial therapeutic effect is obtained only in Schistosoma haematobium infections. In this review on available data of metrifonate it is suggested that further more detailed studies of both S. haematobium and S. mansoni are necessary. This should include studies of the enzymic properties of the worms and the reaction of their esterases towards both metrifonate and DDVP as well as the pharmacokinetics of these compounds in man. In addition there are still unsolved discrepancies reported regarding organ toxicity of the compound which may, however, be due to different grades of parity of the test material.

A method for qualitative and quantitative analysis of trace amounts of quaternary organoarsenicals such as arsenocholine and acetylarsenocholine has been developed. The method is based on pyrolysis, gas chromatography/mass spectrometry and use of deuterium-labelled internal standards. Arsenocholine and acetylarsenocholine have been estimated in fish from arsenic-polluted brackish water and compared with the same species of fish from unpolluted water. The investigation also includes some fish and crustacea from marine water. The presence of arsenocholine and acetylarsenocholine in different aquatic organisms indicate the existence of a general metabolic pathway for these compounds in aquatic ecosystems.

Metrifonate (0,0-dimethyl-[1-hydroxy-2,2,2-trichloroethyl]-phosphonate), and its rearrangement product dichlorvos (2,2-dichlorovinyl dimethyl phosphate) (DDVP), were studied in plasma from two patients with schistosomiasis who were treated with Bilarcil. A mass fragmentographic technique was used. Isotopic variants of the substances were used as internal standards and to compensate for DDVP formed during the workup procedure. The results were related to erythrocyte and plasmacholinesterase determinations. The method described makes it possible to study pharmacokinetics in man and to relate this information to therapeutic effects. It is proposed that metrifonate acts as a slow release formulation for DDVP.

Diazepam has previously been shown to affect the acetylcholine synthesizing system in mouse brain. This paper reports studies on the effect of diazepam on muscarinic receptor density and on pharmacological effects of oxotremorine. The receptor density was studied using a new technique that allows such studies to be performed in vivo under physiological conditions. The method is based on the fact that L-hyoscyamine, the active antipode of atropine, binds specifically to muscarinic receptors in the brain, and can be measured with high sensitivity by gas chromatography-mass spectrometry. Diazepam was found to modify the binding properties of muscarinic receptors in CNS, thereby decreasing the functional receptor pool. It also prevented tremor induced by the muscarinic agonist oxotremorine. Diazepam could however not prevent the hypothermia induced, but rather accentuated this effect of oxotremorine. It is concluded that diazepam, directly or indirectly, influences the effect of cholinergic stimulators by modulating the size of the muscarinic receptor pool.

The four stereoisomers of soman (O-(1,2,2-trimethylpropyl)-methyl-fluorophosphonate) have been analyzed in vivo in mouse blood and tissues after administration of doses corresponding to 0.75 × LD50 of the two diastereoisomeric pairs of soman (S c - and R c -soman). The disappearance of the four isomers has been studied in vitro in the presence of enzymes involved in the toxicity and detoxification of soman, e.g., acetyl- and pseudocholin-esterase, aliesterase, and phosphorylphosphatase. The effect of S c - and R c -soman on brain acetylcholine was studied in the mouse. The analytical methods used are based on gas chromatographymass spectrometry with deuterated internal standards. R c R p - and S c R p -soman, the two isomers that preferentially react with acetylcholinesterase, were found in blood and liver. In liver the concentration of S c R p was higher than that of R c R p and could be followed for 18 hr. In blood only S c R p could be found. Its presence there could be followed during 18 hr. The levels were, however, lower than in liver. The results indicate that the liver might be a depot for soman and that S c R p might be responsible for the delayed intoxication noted after treatment with antidotes. R c -soman was found to have a more pronounced effect on the acetylcholine synthesizing system than has S c -soman, which might explain its higher in vivo toxicity.

Succinylcholine, a bis-quaternary ammonium compound, was extracted from embalmed tissues as an ion-pair with hexanitrodiphenylamine in methylene chloride. The evaporated ion-pair residue is demethylated with sodium benzenethiolate. The tertiary amine formed is identified and quantitated by gas chromatography/mass spectrometry (GC/MS) utilizing a 25 meter glass capillary column coated with SE 52. Identification is accomplished by retention time and mass spectrometry. Quantitation is performed after the addition of deuterated succinylcholine as an internal standard by focusing the mass spectrometer on m/z 58 (for demethylated succinylcholine) and m/z 62 (for the internal standard). The method is applied to the quantitation of succinylcholine from the embalmed kidney, liver, and muscle of rats injected i.m. with 10-200 mg/kg. After six months of storage, the succinylcholine can still be identified and quantitated with highest concentrations found in the muscle injection site. Concentrations as low as 5 ng/g are easily detected.

The effect of diazepam on the acetylcholine (ACh) synthesizing system has been studied in mouse brain in vivo. ACh and choline (Ch) were analyzed by gas chromatography - mass spectrometry using deuterated internal standards. Turnover of ACh was studied by following the incorporation of Ch into ACh after an intravenous injection of [2H6]-Ch. The mice were killed by focussed microwave irradiation on the head. Diazepam was found to increase the endogenous level of Ch, while the concentration of [2H6]-Ch was only half of that of the controls. The incorporation of [2H6]-Ch into [2H6]-ACh was decreased, while the endogenous level of ACh was slightly increased. The turnover rate of ACh was decreased, consistent with a decrease in neuronal excitability induced by diazepam. The elevated endogenous Ch-level and the lower concentration of [2H6]-Ch in the brain, might be explained by an effect of diazepam on the Ch-transport across the blood-brain barrier. This theory is supported by experiments where levels of endogenous and [2H6]-labelled Ch were analyzed in blood following an intravenous injection of [2H6]-Ch. The [2H6]-Ch was found to be eliminated faster in blood from diazepam treated mice. The increased blood level of endogenous Ch, induced by the [2H6]-Ch injection also returned more rapidly to normal in these animals. This is consistent with peripheral Ch being eliminated faster when the central Ch supply is decreased.

The fate of the four stereoisomers of soman [0-(1,2,2- trimethylpropyl )-methyl-fluoro phosphonate] has been studied a) in vivo in mouse blood and liver after IP injection of 0.75 X LD50 RC- and SC-soman respectively, and b) in vitro upon incubation with acetyl- und pseudocholinesterase, aliesterase and phosphorylphosphatase . The analytical method used is based on gas chromatography-mass spectrometry with deuterated internal standard. Most soman disappeared very rapidly from blood and liver. In liver, SCRP and RCRP , the two isomers that preferentially react with cholinesterase, could be detected. The level of SCRP , which was higher than that of RCRP , could be followed for 17-18 h. In blood only SCRP could be detected. The amounts found were fairly constant during the time period 2 min to 4h, and it could even be detected 17-18 h after soman administration.

Metrifonate (0,0-dimethyl-(1-hydroxy-2,2,2-trichloroethyl)-phosphonate) is an organophosphorus compound where there are excellent possibilities to make studies in man. Metrifonate and its rearrangement product dichlorvos (2,2-dichlorovinyl dimethyl phosphate, DDVP) were studied in human blood from schistosomiasis patients treated with Bilarcil®. A mass fragmentographic technique was employed. Deuterium labelled variants of the substances were used, both as internal standards and to compensate for DDVP formed during the workup procedure. The amount of DDVP in plasma was about 1% of the amount of metrifonate. In erythrocytes the corresponding amount of DDVP in percent of metrifonate was half or less. Both compounds reached peak levels within two hours and were detectable for at least eight hours. The results were compared to erythrocyte and plasma cholinesterase determinations. Levels of metrifonate and DDVP, together with cholinesterase activity, have also been studied in mouse brain, liver and kidney. It is proposed that metrifonate acts as a slow release formulation for DDVP. Clearance of metrifonate in man occurs primarily via DDVP. Mild vertigo subsiding in a few hours was the most common side-effect.

The neuromuscular blocking agent succinylcholine (SCh) has been identified and quantitated in biological material using gas chromatography-mass spectrometry. The bisquaternary ammonium compound SCh is extracted from tissue homogenates or body fluids into dichloromethane as an ion pair with hexanitrodiphenylamine (DPA). The evaporated ion pair residue is demethylated with sodium benzenethiolate to form the corresponding tertiary amine which is identified and quantitated by gas chromatography-mass spectrometry using a glass capillary column coated with SE 52. In the quantitative analysis deuterated SCh is used as internal standard. The instrument is focussed on m/z 58 for demethylated SCh and m/z 62 or 64 for the internal standard. Concentrations as low as 5 ng SCh iodide/g tissue or body fluid are easily detected.

A method for the simultaneous quantitation of metrifonate (0,0-dimethyl-(1-hydroxy-2,2,2-trichloroethyl)-phosphonate) and dichlorvos (2,2-dichlorovinyl dimethyl phosphate, DDVP) in human blood has been worked out. It is based upon multiple labelling of the compounds with deuterium and gas phase analysis using the mass spectrometer as a selective detector. The amount of DDVP in plasma is about 1% of the amount of metrifonate. In erythrocytes the corresponding amount of DDVP is 0.5% or less of metrifonate. Both compounds reach peak levels in blood within two hours after oral dosing and are detectable for at least eight hours. Cholinesterase activity in plasma reaches zero levels within 15 min. and remains inhibited for more than eight hours. Red blood cell cholinesterase is inhibited only 60-80%. According to kinetic calculations, clearance of metrifonate occurs primarily via dichlorvos. If dichlorvos is the only active component, which in all likelihood it is, it's slow release may be important in the schistosomicidal effect. Clinical data in seven metrifonate treated patients revealed that mild vertigo subsiding in a few hours was the most common side effect.

The history of succinylcholine (SCh) from 1906 up to date has been well documented by Dorkins (1982). It was introduced as a muscle relaxant by several groups around 1950. Its short lasting effect made it ideal for intubation before surgery. For prolonged muscle relaxation it has been largely superseded by other synthetic compounds. Untoward effects have been noted, such as prolonged apnea, cardiovascular effects and muscle fasciculations. In 1954 SCh was introduced in veterinary medicine as a casting agent for large animals (Hansson and Edlund, 1954; Hansson, 1958).