Comparison of selected skin decontaminant products and regimens against VX in domestic swine.

An anesthetized domestic swine model was used to compare the efficacy and cross-contamination potential of selected skin decontaminant products and regimens against the chemical warfare agent, VX. Animals topically exposed to 2x, 3x or 5x LD(50) VX showed typical signs of organophosphate nerve agent poisoning, including miosis, salivation, mastication, dysrhythmias, and respiratory distress prior to death. Animals were exposed to 5x LD(50) VX and then decontaminated 45 min later with the reactive skin decontamination lotion (RSDL), Fuller's earth (FE), 0.5% hypochlorite, or soapy water. Survival was 100% when the reactive skin decontamination lotion or FE was utilized, although 50% of Fuller's earth-decontaminated animals exhibited serious signs of VX poisoning. Decontamination of VX-treated animals with 0.5% hypochlorite was less effective but also increased survival. Soapy water was ineffective in preventing lethality. Blood cholinesterase levels were not predictive of clinical outcome in decontaminated animals. The potential of "decontaminated" VX in open wounds to cause poisoning was assessed by vigorously mixing 5x LD(50) VX with the test decontaminants for 5 min and then placing the mixture onto a full-thickness skin wound. Soapy water was ineffective in preventing lethality. Although treatment with dry Fuller's earth prevented death and all signs of organophosphate poisoning, a significant proportion of treated animals decontaminated with Fuller's earth in aqueous suspension exhibited serious signs of organophosphate poisoning, suggesting that live agent may be desorbed from Fuller's earth when it is exposed to a liquid environment. Animals treated with reactive skin decontamination lotion or 0.5% hypochlorite-VX mixtures showed no signs of organophosphate poisoning during the 6- h test period.

Swine models in the design of more effective medical countermeasures against organophosphorus poisoning.

Although the three most commonly used large mammal species in the safety assessment of drugs remain the dog, the macaque and the marmoset, swine, especially minipigs, have also been widely used over the years in many toxicological studies. Swine present a number of interesting biological and physiological characteristics. Similarities in skin properties with humans have led to extensive in vitro and in vivo studies. There is a specific interest in cardiovascular research, as well as in anaesthesiology and critical care medicine due to common features of swine and human physiology. Although knowledge of swine brain structure and functions remains incomplete, data does exist. The multiple blood sampling that is necessary in pharmacokinetic and toxicokinetic studies are possible, as well as multiparametric monitoring and interventions with equipment used in human clinical settings. Practicality (handling), scientific (stress reduction) and ethical (invasive monitoring) reasons have led research teams to incorporate anaesthesia into their paradigms which makes the analysis of data increasingly difficult. Although not substantiated by scientific data, the swine appears to have an intermediate position in the scale of public perception between non-human primates and animals commonly referred to as pets (i.e. dogs and cats) and rodents. The benefits of the swine model justify the use of these animals in the design of more effective medical countermeasures against known chemical warfare agents (nerve agents, vesicants and lung damaging agents). Exposure to organophosphorus (OP) pesticides represents a severe health issue in developing countries, while OP intoxication with the more lethal military nerve agents is not only of military concern but also a terrorist threat. Tailoring therapeutic regimens to the reality of OP poisoning is of the utmost importance when little experimental data and sparse human clinical data are available in the decision making process. We will present some of the advantages and disadvantages of the swine model in OP countermeasures elaborating on two examples. First, we will present the issues related to the use of anaesthesia during experimental OP poisoning and second we will show how results from experiments with swine can be integrated into a kinetic-based dynamic model to evaluate oxime efficacy. A better knowledge of OP poisoning in swine (comparative toxicokinetics, pharmacokinetics and biochemistry) is definitely necessary before accepting it as a first choice non-rodent model. However, there exists a large amount of data in the model on anaesthesia and different types of shock favouring their use for evaluation of complex situations such as the anaesthesia of OP poisoned patients and combined injuries.

Lack of a role for creatine phosphate kinase in sulphur mustard-induced cytotoxicity.

Several compounds involved in the creatine phosphate kinase (CPK) pathway were evaluated for their protective effects against the chemical warfare (CW) agent sulphur mustard (HD), in primary chick embryo neuron and first passage human skin keratinocyte cultures. High concentrations of both creatine and creatine phosphate were found to be protective under all culture conditions and increased the LC(50) of HD in both culture systems up to approximately 250%. Little difference was observed in the protective activity of these compounds in undifferentiated versus differentiated neuronal culture, or in proliferating versus differentiating cultures of keratinocytes. The protective effect of these compounds was found to be strictly prophylactic in nature. Although a modest decline in HD half-life was measured in buffer containing creatine phosphate, this did not account for the protective effects of this compound. In contrast to historical literature reporting 90-100% HD-induced CPK inhibition of purified enzyme, less than 30% of CPK activity was found to be inhibited by HD in both human keratinocytes and in swine blood plasma. Incubation of keratinocyte cultures with creatine or creatine phosphate prior to HD exposure did not alter CPK activity, compared with HD-only treated cultures. Although high mM concentrations of both creatine and creatine phosphate exert significant protective effects against HD, these results do not support a role for CPK in its toxicity or in the development of medical countermeasures against this CW agent.

Correlation between formation of a specific hydrocarbon-deoxyribonucleoside adduct and tumor-initiating activity of 7,12-dimethylbenz(a)anthracene and its 9- and 10-monofluoroderivatives in mice.

The formation of epidermal DNA adducts from 9-fluoro-7,12-dimethylbenz(a)anthracene (9-F-DMBA) was compared with 7,12-dimethylbenz(a)anthracene (DMBA) and 10-fluoro-7,12-dimethylbenz(a)anthracene (10-F-DMBA) in SENCAR mice. 9-F-DMBA is equipotent, whereas 10-F-DMBA is more potent than DMBA for skin tumor initiation in this mouse stock. The quantity of covalently bound DNA adducts was essentially identical between 9-F-DMBA and DMBA at all doses tested in the range of 10 to 100 nmol/mouse. These results correlated closely with the dose-response relationships for tumor initiation by the two hydrocarbons. A quantitative comparison of the hydrocarbon-DNA adducts formed after topical application of 100 nmol of DMBA, 9-F-DMBA, and 10-F-DMBA yielded interesting results. The total binding for the three hydrocarbons at this dose was 16.2 +/- 2.6, 18.4 +/- 2.4, and 52.3 +/- 6.8 pmol/mg of epidermal DNA, respectively. Analysis of these DNA adduct samples by dihydroboronate chromatography demonstrated marked reductions in the percentage of syn-diol-epoxide-DNA adducts with both 9-F-DMBA (24%) and 10-F-DMBA (18%) compared with DMBA (57%). Analysis of DNA adduct samples from DMBA-, 9-F-DMBA-, and 10-F-DMBA-treated mice (100 nmol/mouse) by high-pressure liquid chromatography revealed qualitatively similar profiles. However, a quantitative comparison of the three major DNA adducts, tentatively identified as anti-diol-epoxide-deoxyguanosine (Peak I), syn-diol-epoxide-deoxyadenosine (Peak II), and anti-diol-epoxide-deoxyadenosine (Peak III), revealed significant differences. With both 9-F-DMBA and 10-F-DMBA there were marked increases (236% and 644%, respectively) in the quantity of Peak I compared to DMBA. On the other hand, Peak II was formed in approximately equal amounts with DMBA and 10-F-DMBA but only 50% of the DMBA value with 9-F-DMBA. Interestingly, Peak III was formed in approximately equal amounts with both DMBA and 9-F-DMBA but was increased to 337% of the DMBA value with 10-F-DMBA. Thus, the actual level of Peak III (tentatively identified as anti-diol-epoxide-deoxyadenosine) correlated closely with the tumor-initiating activity of these three hydrocarbons, whereas the levels of the other two adducts did not. These data suggest that formation of a specific DNA adduct may be important for DMBA skin tumor initiation. These data are discussed in relation to skin tumor initiation by other hydrocarbons.

Kinetics of formation and disappearance of 7,12-dimethylbenz(a)anthracene:DNA adducts in mouse epidermis.

The rates of formation and disappearance of 7,12-dimethylbenz(a)anthracene (DMBA):DNA adducts were analyzed in the epidermis of SENCAR mice over a 21-day time course. Mice were treated topically with 10 nmol of tritium-labeled DMBA per mouse at various times prior to sacrifice. Under these experimental conditions, total covalent binding of DMBA to epidermal DNA reached a peak at 24 h, and thereafter, DMBA:DNA adduct disappearance was biphasic. The early phase of DMBA:DNA adduct disappearance (Phase A) between 24 and 72 h had a half-life of 3.17 +/- 1.1 days, whereas the later phase (Phase B) had a half-life of 6.46 +/- 1.3 days. A comparison of the biphasic disappearance of total DMBA:DNA adducts with total benzo(a)pyrene:DNA adducts at comparable tumor-initiating doses (i.e., doses producing similar papilloma responses in SENCAR mice) revealed that the half-life for Phase A disappearance of benzo(a)pyrene:DNA adducts was approximately 3 times faster than for DMBA:DNA adducts (1.08 +/- 0.3 days versus 3.17 +/- 1.1 days), respectively. Phase B disappearance of DNA adducts was essentially identical for both hydrocarbons and was similar to the rate of loss of label in epidermal DNA due to cell turnover. The rates of formation and disappearance of the three major DNA adducts derived from DMBA were also examined. Peaks II (syn-diol-epoxide deoxyadenosine) and III (anti-diol-epoxide deoxyadenosine) disappeared more rapidly than Peak I (anti-diol-epoxide deoxyguanosine) beyond 24 h. The data support the conclusion that, for a particular hydrocarbon such as DMBA, deoxyadenosine adducts disappear from epidermal DNA faster than the corresponding deoxyguanosine adducts. In addition, the data suggest that, at the doses used, total DMBA:DNA adducts disappear initially more slowly from epidermal DNA than benzo(a)pyrene:DNA adducts.

Synthesis of the tumorigenic 3,4-dihydrodiol metabolites of dibenz[a,j]anthracene and 7,14-dimethyldibenz[a,j]anthracene.

Syntheses are described of the trans-3,4-dihydrodiol derivatives (2a and 2b) of dibenz[a,j]anthracene and 7,14-dimethyldibenz[a,j]anthracene (1a and 1b), implicated as their proximate carcinogenic metabolites. Conversion of 2a to the bay region anti-diol epoxide derivative 3a, its putative ultimate carcinogenic metabolite, is also reported. The related diol epoxide derivative of 2b could not be prepared due to its chemical instability. Tumorigenicity assays confirm that 1b and 2b are potent carcinogens on mouse skin, while 1a and 2a are only relatively weakly active. The diol epoxide 3a exhibited significantly higher tumorigenicity than its dihydrodiol precursor 2a. These findings are consistent with the hypothesis that the bay region diol epoxide metabolites are the active carcinogenic forms of these hydrocarbons. They also support the generalization that methyl substitution in bay regions enhances the carcinogenic activity of polycyclic aromatic hydrocarbons.

Biologically active dihydrodiol metabolites of polycyclic aromatic hydrocarbons structurally related to the potent carcinogenic hydrocarbon 7,12-dimethylbenz[a]anthracene.

Syntheses of the trans-dihydrodiol derivatives implicated as the proximate carcinogenic metabolites of the polycyclic hydrocarbons cholanthrene, 6-methylcholanthrene, benz[a]anthracene, and 7- and 12-methylbenz[a]anthracene are described. These compounds are useful models for research to determine the molecular basis of the strong enhancement of carcinogenicity consequent upon methyl substitution in nonbenzo bay molecular sites and meso regions of polycyclic hydrocarbons. Synthesis of the bay region anti-diol epoxide derivative of cholanthrene, its putative ultimate carcinogenic metabolite, is also described. Tumorigenicity assays indicate that the 9,10-dihydrodiol derivatives of cholanthrene and its 3- and 6-methyl derivatives are all potent tumor initiators on mouse skin. The most active member of the series is the dihydrodiol derivative of 6-methylcholanthrene, which contains a bay region methyl group. The ability of the dihydrodiols 3a-c and the trans-3,4-dihydrodiol of 7,12-dimethylbenz[a]anthracene (3d) to induce chromosomal aberrations in rat bone marrow cells was also examined. The observed order of activity was 3d greater than 3c greater than 3b greater than 3a. These findings are consistent with the hypothesis that the diol epoxide metabolites of these dihydrodiols are the active carcinogenic forms of the parent hydrocarbons.

Further investigations into the effect of non-benzo ring bay-region methyl substituents on tumor-initiating activity of polycyclic hydrocarbons.

The effect of a methyl substituent at the non-benzo ring bay-region position of benzo[e]pyrene (B[e]P), cholanthrene (CA) and dibenz[a,j]anthracene (DB[a,j]A) on skin tumor-initiating activity was examined. A methyl substituent at the 1-carbon of B[e]P enhanced tumor-initiating activity of the parent compound (0.18 vs. 2.4 papillomas/mouse for B[a]P vs. 1-methyl-B[e]P, respectively, with an 800 nmol initiating dose). A methyl substituent at the 7- and 14-carbons of DB[a,j]A increased the activity of this weak initiator more than 13 times. The introduction of a methyl substituent at the non-benzo ring bay-region position of CA (i.e. carbon atom 6) dramatically increased tumor-initiating activity in SENCAR mice. 6-Methyl-CA was found to be a more potent skin tumor-initiator than 3-methyl-CA, and nearly as potent as 7,12-dimethylbenz[a]anthracene, one of the most potent initiators yet tested in the 2-stage initiation-promotion mouse skin model. When taken together with previous results from our laboratories, the data further support the generality of the enhancing effect of a non-benzo ring bay-region methyl substituent on polycyclic hydrocarbon tumor-initiation.

Competitive binding to the cytosolic 2,3,7,8-tetrachlorodibenzo-p-dioxin receptor. Effects of structure on the affinities of substituted halogenated biphenyls--a QSAR analysis.

The proposed mechanism of action of the toxic halogenated aromatics, typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), involves the initial binding to a high-affinity, low-capacity, cytosolic receptor protein. Previous studies have shown that several 4'-halo-2,3,4,5-tetrachlorobiphenyls bind to the TCDD receptor and that a lateral substituent on both phenyl rings is required for activity. Using an extended series of eighteen 4'-substituted-2,3,4,5-tetrachlorobiphenyls as probes, the effects of a variable lateral substituent on receptor binding affinity and the induction of aryl hydrocarbon hydroxylase (AHH) in vivo and in rat hepatoma H-4-II E cells have been determined. For most substituents, there was an excellent correlation between the rank-order potency for receptor binding and the rank-order potency for AHH induction. Based on in vitro binding affinities (EC50 values) of the 4'-substituted tetrachlorobiphenyls, a multiparameter regression equation was formulated correlating the binding constants to physicochemical substituent parameters. For thirteen compounds out of the present series, multiple regression analysis of the binding data led to the following equation: log(1/EC50) = 1.53 sigma + 1.47 pi + 1.09HB + 4.08, r = 0.978. The results suggest that halogen substitution on both phenyl rings is not a requirement for binding and that hydrophobic (pi) and electronic (sigma) substituent constants and a variable for hydrogen bond (HB) formation are significant parameters describing relative binding avidities of this series of substituted biphenyls for the TCDD receptor.

Modulation of sulfur mustard toxicity by arginine analogues and related nitric oxide synthase inhibitors in vitro.

The modulating effects of a series of arginine analogues and related nitric oxide synthase inhibitors against the toxicity of sulfur mustard (HD) in primary cultures of chick embryo forebrain neurons were examined. In addition to the previously identified protective compounds, D- and L-nitroarginine methyl ester, eight additional arginine analogues were shown to have significant, concentration-dependent protective characteristics against HD toxicity. Of these, L-nitroarginine was the most potent, increasing the LC50 of vehicle-pretreated HD-treated control cultures by approximately 350%. In addition to these protective agents, five compounds related to arginine were also identified that potentiated the toxicity of HD in the neuron cultures in a concentration-dependent manner. This action occurred at concentrations where these chemicals alone exhibited no toxicity. Characterization of the active compounds in this study showed that it was likely that the protective agents, as well as those compounds that potentiated HD toxicity, were exerting their effects at the same biochemical target, but not through the inhibition of nitric oxide synthase. Although the identity of this target site is as yet unknown, these studies demonstrate that subtle alterations to the arginine structure can yield compounds that differentially modulate the toxicity of HD through their activity at a common target site.

Parallel development of acetylcholinesterase in vivo and in primary neuron surface culture.

The development of acetylcholinesterase (AChE) activity in primary surface cultures of mouse cortical neurons and in mouse brain was examined. The specific activity of AChE in culture increased over 600% during a 3 week period and closely paralleled the development of AChE observed in vivo. The results obtained in this study show that a developmental increase in AChE can be obtained in primary surface neuron cultures, and that the high degree of cellular organization previously deemed necessary for this development in vitro is not as important as previously thought.

Characterization of the protective effects of L-nitroarginine methyl ester (L-NAME) against the toxicity of sulphur mustard in vitro.

The protective effects of L-nitroarginine methyl ester (L-NAME) against the toxicity of sulphur mustard (HD) was characterized in primary cultures of chick embryo forebrain neurons. These effects were not associated with the known nitric oxide synthase-inhibiting characteristics of this compound. No protection was evident in immature (1 day old) cultures. However, L-NAME pre-treatment resulted in a concentration-dependent protection against the toxicity of HD in mature (5 days and older) cultures, with maximal protection reaching greater than 220% at 5 mM L-NAME in terms of increasing the LC50 of control HD-treated cultures. Maximal protective effects were also achieved when L-NAME treatment was delayed up to 3 h post-HD exposure. These effects were reduced by 5 h and absent when the L-NAME was added to the cultures 8 h after HD exposure. Protection against the toxicity of HD was dependent on the continued presence of L-NAME in the medium and was persistent up to 48 h after HD exposure. This compound is one of the most effective drugs yet identified in protecting against the toxicity of HD and is the only one that exerts its effects therapeutically.

Effect of lowered temperature on the toxicity of sulphur mustard in vitro and in vivo.

Primary cultures of chick embryo neurons were exposed to sulphur mustard (HD) and L-nitroarginine methyl ester (L-NAME) and then incubated at either 25 or 37 degrees C. Lowering the temperature of the cultures decreased the 24-h toxicity of HD, but did not increase the efficacy of L-NAME protection. However, the length of time post-HD treatment in which L-NAME was maximally effective in protecting against HD toxicity was dramatically enhanced, out to 12 h after HD exposure. In addition, the persistence of L-NAME protection of the cells against HD was significantly lengthened. Tests conducted in human skin keratinocytes also showed that lowering the incubation temperature of actively proliferating, just-confluent or post-confluent cultures significantly and persistently decreased the cytotoxicity of HD. The persistence of L-NAME protection was increased in non-proliferating cells. Finally, cooling of HD-vapour exposed sites on hairless guinea pigs for 4.5 h decreased the severity of the resultant lesions out to 72 h post-exposure.

Toxicity of sulphur mustard in adult rat lung organ culture.

The toxicity of the chemical warfare agent sulphur mustard, (bis-(2-chloroethyl)sulphide, HD), was examined in adult rat lung organ cultures. Assessment of HD-induced damage by the MTT cytotoxicity assay indicated that the median lethal concentration (LC50) of HD in these cultures was reproducible, and in the microM range. Damage to the lung slices was expressed only after a latent period of 48 h and did not increase significantly with longer expression times. Histopathological examination of HD-treated lung cultures showed that the structural changes in the lung tissue paralleled the toxicity measured biochemically, and were also similar to the damage found in animals and man exposed to HD in vivo. This in vitro model offers a useful tool with which to study the toxicity and mechanism of action of sulphur mustard.

Efficacy of an oximate-based skin decontaminant against organophosphate nerve agents determined in vivo and in vitro.

Recent Canadian research efforts have been directed towards the development of a reactive skin decontaminant (RSD) lotion active against classical nerve agents and mustard. The formulation presently under study consists of a 1.25 molal solution of potassium 2,3-butanedione monoximate (KBDO) in polyethylene glycol methylether 550. Although this formulation has shown good efficacy, concern has been expressed as to the potential toxicity of the reaction products of KBDO and organophosphate (OP) nerve agents. This report details the high efficacy of this lotion in inactivating OPs as measured by the systemic toxicity of the OP/RSD mixtures in rats. In addition, primary cultures of chick embryo neurons were also used to test the efficacy of the RSD. By relating the anticholinesterase activity in these cultures of the OP/RSD mixture to that of pure OP standards, a sensitive measure of the value of the RSD in inactivating tabun, sarin, soman and VX was obtained. Experiments with all four nerve agents in this in vitro system provided a good correlation with the in vivo data, and also indicated that the inactivation process was time- and agent-dependent and also related to the ratio of OP to RSD.

Aryl hydrocarbon hydroxylase (AHH) induction by polybrominated biphenyls (PBBs): enchancement by photolysis.

Irradiation of the commercial polybrominated biphenyl (PBB( mixture, fireMaster BP-6, in cyclohexane solution at 300 nm for 930 min resulted in a marked diminution of the major components of the mixture. Administration of the photolyzed PBB mixture of fireMaster BP-6 to immature male Wistar rats caused both dose-related decreases in thymus weight and increase in hepatic microsomal benzo[a]pyrene hydroxylase (AHH), 4-dimethylaminoantipyrine N-demethylase and NADPH-cytochrome c reductase activities and cytochrome P-450 content. The dose effecting half-maximal AHH induction for the photolyzed PBBs (9 mg . KG-1) was approximately 6 times lower than that of fireMaster BP-6 (50 mg. kg-1). Furthermore, the concentration of photolyzed PBBs (2 micrometers) required to displace 50% of the specifically-bound [3H] TCDD from its high-affinity cytosolic Ah receptor was approximately 150 times lower than that required for fireMaster BP-6 (300 micrometers), as measured by sucrose density gradient centrifugation analysis. The results suggest that the photolysis of the commercial PBB mixture yields products which possess increased biologic activity.

Toxicity of sulfur mustard in primary neuron culture.

The toxicity of sulfur mustard (HD) was assessed in primary cultures of chick embryo forebrain neurons using several different endpoints. Mature neurons were found to be very sensitive to the toxic effects of this agent and tritiated arachidonic acid release, as well as the MTT, neutral red and alamarBlue cytotoxicity assays all gave LC(50) values in the low mum range. Maximal toxicity was initiated within minutes of culture exposure to HD and was not found to be associated with toxic mediator release into the medium. The characteristics of toxicity were quite different when comparing immature cultures to mature ones. Mature cultures were more sensitive to the toxicity of HD than were immature cultures, and maximal toxicity in mature cultures took longer to be expressed. In addition, the toxicity was found to be dependent on the initial seeding density, as well as on the age of the cultures at the time of chemical treatment. Although the reasons for these observations are unclear, the apparent dependence of HD toxicity on the differentiative maturity of the cultures may eventually provide some clues as to the mechanism of action of this chemical agent. Furthermore, the extreme sensitivity of these cells to the toxic effects of HD makes them a useful model system with which to screen for potential protective drug regimens against this chemical warfare agent.

Cytotoxicity of the MEIC test chemicals in primary neurone cultures.

50 Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) test chemicals were assayed for cytotoxicity in primary cultures of chick embryo forebrain neurones using the MTT and neutral red assays. The neutral red assay was consistently more sensitive to chemical toxicity; however, both assays were equally predictive when compared with in vivo toxicity data obtained from the Registry of Toxic Effects of Chemical Substances. High correlations were obtained when comparing the in vitro data with ip rodent toxicity data, but these correlations decreased significantly when oral toxicity data were used. The predictive value of the in vitro data for oral human toxicity was generally poor, but comparable with its value in predicting oral rodent toxicity. In a limited study with 10 of the MEIC test chemicals, the cytotoxicity of some compounds was dependent on the degree of differentiation of the neurone cultures, suggesting that this culture system may not only be sensitive to the basal cytotoxicity of chemicals, but also to toxic effects specific to the specialized differentiated functions of the central nervous system.

Effect of intracellular calcium modulation on sulfur mustard cytotoxicity in cultured human neonatal keratinocytes.

Previous studies in human skin keratinocyte cultures have shown that sulfur mustard (HD) induces an immediate and irreversible increase in internal free calcium levels that was independent of external calcium concentrations. These findings suggested a role for calcium in the aetiology of HD-induced cell death and that modulation of intracellular calcium concentrations may assist in providing protection against this agent. In the current work, actively proliferating and confluent cultures of first passage neonatal human skin keratinocytes were used to assess the effect of altered intra- and extracellular calcium levels on HD toxicity. Treatment of cultures with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin, or the calcium chelator BAPTA-AM, which reduce HD-induced elevation of intracellular free calcium, did not modulate the toxicity of HD. Furthermore, alteration of external calcium concentrations during these same experiments failed to elicit any change in the viability of HD-exposed cells. Treatment of confluent cultures with ionomycin at either low (100 microM) or high (1.2 mM) external calcium concentrations also failed to modulate the toxicity of HD in any way. It appears that in neonatal human skin keratinocytes in culture, HD-induced intracellular calcium perturbation does not play a major role in HD-induced cytotoxicity.

Effect of metabolism on the anticholinesterase activity of parathion and paraoxon in primary neuron cultures.

The anticholinesterase activity of parathion and paraoxon, and the effects of metabolism on this activity, were examined in primary cultures of chick embryo forebrain neurons. Paraoxon was an extremely potent inhibitor of acetylcholinesterase activity, with a median inhibitory concentration (IC(50)) more than 800 times more potent than parathion. Incubation of these two compounds with rat hepatic S-9 fractions, before neuron treatment, dramatically altered their activity, though with opposite effects. Parathion, when preincubated with control S-9 (from saline-treated rats), exhibited increased anticholinesterase activity, whereas paraoxon was much decreased in potency. The relative anticholinesterase activity of these compounds after incubation with the control S-9 fractions was much more predictive of the relative toxicites of these compounds in vivo. Preincubation of these compounds with hepatic S-9 fractions that had been obtained from rats induced with either phenobarbitone or 3-methylcholanthrene, resulted in a decreased activation of parathion and an increased deactivation of paraoxon compared with control S-9.