Enhanced immunogenicity of line 10 guinea pig hepatocarcinoma cells after culture.

Line 10 hepatocarcinoma cells derived from ascites in a strain 2 guinea pig were tumorigenic when transferred intradermally. After they had been cultured in vitro for 20 days or more in medium enriched with 10% fetal bovine serum (FBS), they became immunogenic. Injections of immunogenic cells did not cause lethal tumors, and recipients were resistant to subsequent challenges with tumorigenic line 10 cells. Resistance was specific since growth of line 1 cells, a syngeneic but antigenically distinct tumor, was not affected. Cells cultured in medium enriched with 10% calf bovine serum or 10% normal guinea pig serum or in reduced concentrations of FBS were less effective in inducing resistance. When cultured line 10 cells were injected ip into normal guinea pigs, ascites tumors developed that were tumorigenic. The growth rate of line 10 cells in culture was considerably decreased as determined by reduced [3H]thymidine incorporation and mitotic indices. The mechanism(s) responsible for enhancement of immunogenicity in cultured line 10 cells is discussed but was not determined.

Genetic and biochemical characterization of the CHO-UV-1 mutant defective in postreplication recovery of DNA.

The CHO-UV-1 mutant, a Chinese hamster ovary cell with defective postreplication recovery of DNA, is 2- to 4-fold more sensitive than its wild-type counterpart (CHO-77256) to the lethal effects of ethylating agents and UV radiation; it is also hypersensitive (10- to 20-fold) to some DNA-methylating and -cross-linking agents. We studied the CHO-UV-1 mutant further to define its phenotype in terms of DNA damage induction and repair, methyltransferase activity, and effects of caffeine on mutational and lethal responses. Both wild-type and CHO-UV-1 cells incurred similar levels and types of damage when exposed to UV radiation, N-methyl-N'-nitro-N-nitrosoguanidine, or N-methyl-N-nitrosourea. The rate and extent of repair of Micrococcus luteus endonuclease-sensitive sites after UV irradiation or treatment with N-methyl-N'-nitro-N-nitrosoguanidine were also equivalent in these two cell types. Twenty % of the initial endonuclease-sensitive sites induced in either cell line remained at 18 h after UV irradiation; approximately 8% of the sites after N-methyl-N'-nitro-N-nitrosoguanidine exposure were present in both parental and CHO-UV-1 cells after a 17-h repair period. Moreover, the ability of CHO-UV-1 to resynthesize and ligate DNA during excision repair was similar to that of its parent. Neither CHO-UV-1 nor CHO-77256 had appreciable levels of O6-methylguanine-DNA methyltransferase activity which ameliorates the cytotoxicity of alkylating agents. Caffeine, a known inhibitor of postreplication repair, decreased the frequency of mutation induction at the hypoxanthine-guanine phosphoribosyltransferase locus by 40-55% in CHO-77256 but not in CHO-UV-1. These results rule out defective excision repair as a factor in the hypersensitivity of the CHO-UV-1 mutant to DNA-damaging agents. Hence, this cell line appears to derive from a mutation affecting nonexcision repair processes and should be useful in clarifying the mechanism(s) of postreplication recovery of DNA in mammalian cells.

Comparison of two in vitro activation systems for protoxicant organophosphorous esterase inhibitors.

In order to perform in vitro testing of esterase inhibition caused by organophosphorous (OP) protoxicants, simple, reliable methods are needed to convert protoxicants to their esterase-inhibiting forms. Incubation of parathion or chlorpyrifos with 0.05% bromine solution or uninduced rat liver microsomes (RLM) resulted in production of the corresponding oxygen analogs of these OP compounds and markedly increased esterase inhibition in SH-SY5Y human neuroblastoma cells. Neither activation system affected cell viability or the activity of AChE or NTE in the absence of OP compounds. Although parathion and chlorpyrifos were activated by RLM, bromine activation required fewer steps and produced more esterase inhibition for a given concentration of chlorpyrifos. However, RLM activation of OP protoxicants produced metabolites other than oxygen analogs and may, therefore, be more relevant as a surrogate for OP biotransformation in vivo. This methodology makes the use of intact cells for in vitro testing of esterase inhibition caused by protoxicant organophosphate compounds a viable alternative to in vivo tests.

Evaluation of knit glove fabrics as barriers to dermal absorption of organophosphorus insecticides using an in vitro test system.

Cotton and synthetic knit glove fabrics in combination with an in vitro skin model were used to examine the capability of fabric to decrease the dermal absorption of the organophosphorus insecticides azinphos-methyl, paraoxon, and malathion. Capability for inhibition of acetylcholinesterase was determined in samples of media taken from under the skin barrier after the skin model, with or without fabric protection, had been exposed to the test compounds for 4 h. Acetylcholinesterase inhibitions caused by the direct addition of organophosphorus insecticide to the media were also included in the comparison. Results indicated that the skin model system alone had some capability to serve as a barrier to the transfer of organophosphates. Fabric covering used on the test model increased the barrier between insecticide application and resultant acetylcholinesterase inhibition. The all-cotton, 7-cut knit was especially effective in preventing the absorption of azinphos-methyl, as this organophosphorus insecticide had no capability to cause acetylcholinesterase inhibition when this fabric was used to protect the skin model. Knit glove materials of 100% cotton were demonstrated to be effective in preventing the absorption of paraoxon and malathion. These studies indicate that an in vitro model system can be used in combination with fabrics to study the relationship between clothing and skin as barriers to the absorption of organophosphorus insecticides.

Neuropathy target esterase inhibition by organophosphorus esters in human neuroblastoma cells.

Certain organophosphorus compounds (OPs) produce a delayed neuropathy (OPIDN) in man and some animal species. Capability to cause OPIDN is generally predicted in animal models by early and irreversible inhibition of neuropathy target esterase (NTE, neurotoxic esterase). In this study, NTE inhibition in response to OP exposure was examined in cell culture, using the human SH-SY5Y neuroblastoma cell line. Cells were exposed for 1 hr to equimolar (1 x 10(-5) M) concentrations of 6 OPs associated with OPIDN in vivo (including 2 protoxicants and 4 active (-P = O) toxicants), and 8 OPs that do not produce delayed neuropathy in animal models (including 5 protoxicants and 3 -P = O compounds). The -P = O compounds that cause OPIDN in animal models inhibited NTE > 60% at the test concentration; -P = O compounds that do not cause OPIDN in animal models inhibited NTE < 30%. Protoxicants did not inhibit NTE at the test concentration, reflecting their limited metabolism in the human cell line. These results indicate that human neuroblastoma cells have potential use in the initial screening of bioactive OPs with capability for causing OPIDN.

Comparative sensitivities of avian neural esterases to in vitro inhibition by organophosphorus compounds.

The inhibitory power of organophosphorus compounds in vitro was compared against neurotoxic esterase (also known as neuropathy target esterase, NTE), acetylcholinesterase and carboxylesterase activities in brains from chickens, turkeys, quail and rats. Brains from the species most susceptible to clinical signs of organophosphorus-induced delayed neuropathy (chicken, turkey) contained more NTE than did rat and quail. Higher concentrations of organophosphorus compounds were required to inhibit rat NTE and quail acetylcholinesterase than were necessary for inhibition of these enzymes in chicken and turkey brains. Total carboxylesterase and acetylcholinesterase activities were less in rats than in the avian species.

Inhibition of carboxylesterases in SH-SY5Y human and NB41A3 mouse neuroblastoma cells by organophosphorus esters.

Carboxylesterases (CbxE) can be inhibited by organophosphorus esters (OPs) without causing clinical evidence of toxicity. CbxE are thought to protect the critical enzyme acetylcholinesterase (AChE) from OP inhibition in animals. CbxE and AChE are both present in neuroblastoma cells, but, even though these cells have potential to be an in vitro model of OP toxicity, the effect of OPs on CbxE and the relationship of CbxE inhibition and AChE inhibition have not yet been examined in these cells. Therefore, this study examined concentration-related OP-induced inhibition of CbxE in human SH-SY5Y and mouse NB41A3 neuroblastoma cells with 11 active esterase inhibitors: paraoxon, malaoxon, chlorpyrifos-oxon, tolyl saligenin phosphate (TSP), phenyl saligenin phosphate (PSP), diisopropyl phosphorofluoridate (DFP), mipafox, dichlorvos, trichlorfon, dibutyryl dichlorovinyl phosphate (DBVP), and dioctyl dichlorovinyl phosphate (DOVP). All could inhibit CbxE, although the enzyme was less likely to be inhibited than AChE following exposure to 9 of the test compounds in the human cell line and to all 11 of the test compounds in the murine cell line. Species differences in concentration-related inhibitions of CbxE were evident. When cells were exposed first to an OP with a low IC50 toward CbxE (PSP), followed by an OP with high affinity for AChE (paraoxon or malaoxon), inhibitions of CbxE and AChE were additive. This indicated that CbxE did not protect AChE from OP-induced inhibition in this cell culture model.

Comparative effectiveness of organophosphorus protoxicant activating systems in neuroblastoma cells and brain homogenates.

The ability of bromine and rat liver microsomes (RLM) to convert organophosphorus (OP) protoxicants to esterase inhibitors was determined by measuring acetylcholinesterase (AChE) and neuropathy target esterase (NTE) inhibition. Species specific differences in susceptibility to esterase inhibition were determined by comparing the extent of esterase inhibition observed in human neuroblastoma cells and hen, bovine, and rodent brain homogenates. OP protoxicants examined included tri-o-tolyl phosphate (TOTP), O-ethyl O-p-nitrophenyl phenylphosphonothioate (EPN), leptophos, fenitrothion, fenthion, and malathion. Bromine activation resulted in greater AChE inhibition than that produced by RLM activation for equivalent concentrations of fenitrothion, malathion, and EPN. For EPN and leptophos, bromine activation resulted in greater inhibition of NTE than RLM. Only preincubation with RLM activated TOTP; resultant inhibition of AChE was less in hen brain (13 +/- 3%) than in neuroblastoma cells (73 +/- 1%) at 10(-6) M. In contrast, 10(-6) M RLM-activated TOTP produced more inhibition of hen brain NTE (89 +/- 6%) than NTE of human neuroblastoma cells (72 +/- 7%). Human neuroblastoma cells and brain homogenates from hens, the accepted animal model for study of OP-induced neurotoxicity, were relatively similar in sensitivity to esterase inhibition. Homogenates from hens were more sensitive to NTE inhibition induced by phenyl saligenin phosphate (PSP), an active congener of TOTP, than were homogenates from less susceptible species (mouse, rat, bovine). AChE of hen brain homogenates was also more sensitive than homogenates from other species to malaoxon, the active form of malathion.

A microassay method for neurotoxic esterase determinations.

A microtiter plate reader with an associated computer to average triplicate samples and subtract blanks was used for reading and calculating neurotoxic esterase (NTE, also known as neuropathy target esterase) activities in spinal cord regions of hens 4 hr after administration of diisopropylphosphorofluoridate (DFP, 0.5 mg/kg sc). Although NTE inhibition is an early indicator of organophosphorus ester-induced delayed neuropathy. DFP-induced inhibition was not greater in regions of the spinal cord where pathological changes are most notable. Acetylcholinesterase (AChE) activities and protein determinations were also done on these tissues using microassay methods. DFP-induced AChE inhibition was similar to NTE inhibition. In addition to the capability to be used for small regional esterase activity measurements, the microassay was advantageous because the number of samples incorporated into a single assay was increased and the time needed for the NTE assay was reduced by 50%. Total volume of incubate in each well was 0.3 ml; the incubate contained 1/20 quantities of sample and reagents necessary in more conventional assays. Validation of the microassay was performed by comparison with more conventional assays when measuring inhibition of NTE and AChE in brains of control and experimental hens of two different genetic strains (B13B13 and B21B21 white leghorns). Experimental birds were given DFP, 0.5 mg/kg sc, 24 hr before samples were collected. NTE activities in brains of control hens were similar using both types of NTE analytical procedures. Percentage inhibition of NTE caused by DFP was within 4% using both assay procedures in both strains of hens.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase and neuropathy target esterase inhibitions in neuroblastoma cells to distinguish organophosphorus compounds causing acute and delayed neurotoxicity.

The differential inhibition of the target esterases acetylcholinesterase (AChE) and neuropathy target esterase (NTE, neurotoxic esterase) by organophosphorus compounds (OPs) is followed by distinct neurological consequences in exposed subjects. The present study demonstrates that neuroblastoma cell lines (human SH-SY5Y and murine NB41A3) can be used to differentiate between neuropathic OPs (i.e., those inhibiting NTE and causing organophosphorus-induced delayed neuropathy) and acutely neurotoxic OPs (i.e., those highly capable of inhibiting AChE). In these experiments, concentration-response data indicated that the capability to inhibit AChE was over 100x greater than the capability to inhibit NTE for acutely toxic, nonneuropathic OPs (e.g., paraoxon and malaoxon) in both cell lines. Inhibition of AChE was greater than inhibition of NTE, without overlap of the concentration-response curves, for OPs which are more likely to cause acute, rather than delayed, neurotoxic effects in vivo (e.g., chlorpyrifos-oxon, dichlorvos, and trichlorfon). In contrast, concentrations inhibiting AChE and NTE overlapped for neuropathy-causing OPs. For example, apparent IC50 values for NTE inhibition were less than 9.6-fold the apparent IC50 values for AChE inhibition when cells were exposed to the neuropathy-inducing OPs diisopropyl phosphorofluoridate, cyclic tolyl saligenin phosphate, phenyl saligenin phosphate, mipafox, dibutyl dichlorovinyl phosphate, and di-octyl-dichlorovinyl phosphate. In all cases, esterase inhibition occurred at lower concentrations than those needed for cytoxicity. These results suggest that either mouse or human neuroblastoma cell lines can be considered useful in vitro models to distinguish esterase-inhibiting OP neurotoxicants.

Effect of laundering on ability of glove fabrics to decrease the penetration of organophosphate insecticides through in vitro epidermal systems.

Two knit glove fabrics, one of 100% cotton and one of 100% polypropylene, were examined for their capability to decrease the penetration of the organophosphate insecticides (OPs), azinphos-methyl and paraoxon after 4 h at field concentrations (3000 and 15 ppm, respectively) through an in vitro epidermal system (Skin2, Advanced Tissue Systems, LaJolla, CA). The glove fabrics were examined under three different conditions of use: new, after they had been abraded and after they had been abraded and then laundered. New and laundered cotton fabric was also examined for its capability to decrease the penetration of azinphos-methyl through another in vitro epidermal system (Epiderm, MatTek Corp., Ashland, MA), after 4 and 24 h of exposure. Capability of the media under the in vitro epidermal systems to inhibit brain acetylcholinesterase (AChE) was used as the indicator of penetration. Results were compared to OP-caused inhibitions seen in media under the fabric alone and in media under the in vitro epidermal systems alone. Incubations of azinphos-methyl suspensions and the in vitro epidermal systems covered with fabric indicated that both the epidermal cells and fabric provided protection against AChE inhibition caused by this OP and that the protective effects were additive, whether measured after 4 or 24 h of exposure. Therefore, neither laundering nor abrasion followed by laundering altered the capability of the in vitro epidermal systems to absorb azinphos-methyl suspension. For paraoxon solution, however, new cotton glove fabric prevented absorption, and this protective effect, noted after 4 h of exposure, was lost when the fabric was laundered. Abrading the fabric did not cause a greater effect than laundering alone. These results suggest that the pesticide as well as its formulation may be factors of consideration when protective fabrics are chosen, and that, for cotton glove fabric, the protection against some OPs may best be provided before the fabric is laundered.

Effect of beta-naphthoflavone on o-tolyl saligenin phosphate-induced delayed neuropathy in two lines of chickens.

The effect of the microsomal enzyme inducer beta-naphthoflavone (beta NF) on the development of organophosphorus-induced delayed neuropathy (OPIDN) was examined in two laboratories (VPI and MSU), utilizing two strains of White Leghorn hens. A single intraperitoneal injection of beta NF at 80 mg/kg body weight 48 h prior to administration of o-tolyl saligenin phosphate (TSP), the neuroactive metabolite of tri-o-tolyl phosphate (TOTP), caused a significant increase in hepatic microsomal cytochrome P-450 concentrations and aniline hydroxylase activities after 72 h in both strains. Hepatic carboxylesterase and cholinesterase activities were not affected by beta NF treatment in either strain. Administration of TSP in single subcutaneous doses of 20 and 25 mg/kg body weight (VPI) or 30 and 60 mg/kg body weight (MSU) caused significant inhibition of whole-brain neuropathy target esterase (NTE) activity 24 h postdosing, and hens subsequently developed clinical signs characteristics of OPIDN. beta NF had no significant effect on NTE inhibition or on initiation or severity of OPIDN clinical signs. However, OPIDN clinical signs were less severe in the strain of bird (MSU) with the higher intrinsic hepatic carboxylesterase activity and the higher beta NF-induced cytochrome P-450 concentration. The study indicates that microsomal enzyme induction, which has been shown to alleviate TOTP-induced delayed neuropathy, could not alleviate OPIDN resulting from exposure to TSP. This study also suggests that strain may affect susceptibility to TSP-induced delayed neuropathy.

Regulation of transcription by cyclic AMP-dependent protein kinase.

cAMP-dependent protein kinase (PKA; ATP: protein phosphotransferase; EC 2.7.1.37) appears to be the major mediator of cAMP responses in mammalian cells. We have investigated the role of PKA subunits in the regulation of specific genes in response to cAMP by cotransfection of wild-type or mutant subunits of PKA together with cAMP-inducible reporter genes. Overexpression of catalytic subunit induced expression from three cAMP-regulated promoters (alpha-subunit, c-fos, E1A) in the absence of elevated levels of cAMP but did not affect expression from two unregulated promoters (Rous sarcoma virus, simian virus 40). Cotransfection of a regulatory subunit gene containing mutations in both cAMP binding sites strongly repressed both basal and induced expression from the cAMP-responsive alpha-subunit promoter without affecting expression from the Rous sarcoma virus promoter. These experiments indicate that cAMP induces gene expression through phosphorylation by the catalytic subunit and that the ambient degree of phosphorylation dictates the level of basal as well as induced expression of the cAMP-regulated alpha-subunit gene.

Expression and characterization of mutant forms of the type I regulatory subunit of cAMP-dependent protein kinase. The effect of defective cAMP binding on holoenzyme activation.

The mouse wild type and four mutant regulatory type I (RI) subunits were expressed in Escherichia coli and subjected to kinetic analyses. The defective RI subunits had point mutations in either cAMP-binding site A (G200/E), site B (G324/D, R332/H), or in both binding sites. In addition, a truncated form of RI which lacked the entire cAMP-binding site B was generated. All of the mutant RI subunits which bound [3H]cAMP demonstrated more rapid rates of cAMP dissociation compared to the wild type RI subunit. Dissociation profiles showed only a single dissociation component, suggesting that a single nonmutated binding site was functional. The mutant RI subunits associated with purified native catalytic subunit to form chromatographically separable holoenzyme complexes in which catalytic activity was suppressed. Each of these holoenzymes could be activated but showed varying degrees of cAMP responsiveness with apparent Ka values ranging from 40 nM to greater than 5 microM. The extent to which the mutated cAMP-binding sites were defective was also shown by the resistance of the respective holoenzymes to activation by cAMP analogs selective for the mutated binding sites. Kinetic results support the conclusions that 1) Gly-200 of cAMP-binding site A and Gly-324 or Arg-332 of site B are essential to normal conformation and function, 2) activation of type I cAMP-dependent protein kinase requires that only one of the cAMP-binding sites be functional, 3) mutational inactivation of site B (slow exchange) has a much more drastic effect than that of site A on increasing the Ka of the holoenzyme for cAMP, as well as in altering the rate of cAMP dissociation from the remaining site of the free RI subunit. The strong dependence of one cAMP-binding site on the integrity of the other site suggests a tight association between the two sites.

Toxicity and toxicokinetics of carbaryl in chickens and rats: a comparative study.

Carbaryl, a carbamate insecticide, exerts its toxic effect in animals by inhibiting the activity of neural acetylcholinesterase. Differences in sensitivity of this enzyme to inhibition were studied after intraperitoneal administration to chickens and rats. A dose of 900 mg/kg to chickens and 70 mg/kg to rats caused equivalent inhibition of brain cholinesterase activities (57% +/- 6 and 47% +/- 4, respectively) 60 min after administration, which was the time of maximal cholinergic signs. Signs of toxicity (salivation, respiratory distress, muscle tremors and weakness) were more pronounced in rats than in chickens when brain acetylcholinesterase was inhibited to the same extent in both species. Carboxylesterase activities in brain, liver, and plasma were also inhibited 60 min after administration of carbaryl to chickens and rats. Activities of enzymes associated with hepatic microsomes were unaffected. Specific activities of brain esterases, including acetylcholinesterase, carboxylesterase and neurotoxic esterase, were higher in untreated chickens than in untreated rats. Specific activities of liver esterases (carboxylesterase, A-esterase) were, however, 4- and 10-fold lower in untreated chickens than in untreated rats. Total clearance of carbaryl in the chicken, determined after intravenous administration of 5 mg/kg, was 0.26 +/- 0.02 l/kg/min. This value is 5.7 times higher than that reported for the rat, indicating that the relatively lower activities of esterases in the liver of chickens did not affect the clearance of this chemical in the avian species.

Inhibition of intracellular cAMP-dependent protein kinase using mutant genes of the regulatory type I subunit.

Expression vectors were constructed that code for mutated forms of the regulatory type 1 subunit (RI) of cyclic AMP-dependent protein kinase. These mutations alter a specific amino acid which is present in each of two homologous cAMP-binding domains of the RI protein. When these expression vectors were introduced into NIH 3T3 and Y1 adrenocortical tumor cells a mutant RI protein was produced that acted in a dominant fashion to cause a 20-400-fold inhibition of cAMP-dependent protein kinase activation. In addition, processes controlled by cAMP in adrenal cells were blocked; cells became resistant to the growth-inhibitory effects of cAMP and defective in steroid synthesis. Expression of mutant RI genes in cells provides a specific means to explore the role of cAMP and protein phosphorylation in the process of intracellular signalling.

Catecholamine concentrations and contractile responses of isolated vessels from hens treated with cyclic phenyl saligenin phosphate or paraoxon in the presence or absence of verapamil.

Blood samples and vascular segments from the ischiadic artery of hens treated with either cyclic phenyl saligenin phosphate (PSP; 2.5 micrograms/kg, im) or paraoxon (PXN; 0.1 micrograms/kg, im) in the presence or absence of verapamil, a calcium channel antagonist (7 micrograms/kg, im, given 4 consecutive days beginning the day before PSP or PXN administration), were examined 1, 3, 7, and 21 d after PSP or PXN administration in order to determine the contribution of catecholamines and peripheral blood vessel physiology and morphology to organophosphorus-induced delayed neuropathy (OPIDN). The levels of plasma catecholamines were measured by high-performance liquid chromatograpy (HPLC) and indicated a different effect with PSP, which causes OPIDN, and PXN, which does not. PSP treatment elevated the levels of norepinephrine and epinephrine throughout the study, while PXN treatment depressed the levels of these catecholamines. Verapamil treatment attenuated the OP response by approximately 50% for both compounds. Ischiadic vessel segments were isolated from OP-treated hens and perfused at a constant flow rate of 12 ml/min, then examined for their response to potassium chloride (KCl, 3 x 10(-3) M), acetylcholine (ACh), phenylephrine (PE), an alpha 1 adrenergic agonist, and salbutamol (SAL), a beta 2 adrenergic agonist. Agents were delivered in concentrations of 10(-8) to 10(-3) M. Vascular segments did not respond to ACh or SAL at any concentration used. Vessels displayed a significant reduction in contractile response to both KCl (3 x 10(-3) M) and PE (10(-8) to 10(-3) M) 3 and 21 d after exposure to either PSP or PXN. This reduced response was not altered by the presence of verapamil. Innervation of the peripheral vasculature was unchanged after OP treatment. This study indicates that plasma catecholamine levels could be differentially altered by treatment with OPs that do and do not cause OPIDN and suggests that the alterations involve intracellular calcium. In contrast, vascular response of the ischiadic artery was altered following OP treatment, but the effect was not specific for the neuropathy-inducing OP, PSP, and response was not mediated by Ca 2+, nor was it the result of autonomic nerve deterioration.

Protease activity in brain, nerve, and muscle of hens given neuropathy-inducing organophosphates and a calcium channel blocker.

Activity of calcium-activated neutral protease (CANP or calpain), an enzyme responsible for degradation of axonal and muscle cytoskeletal elements, was determined in brain, sciatic nerve, and gastrocnemius muscle of hens given tri-ortho-tolyl phosphate (TOTP, 360 mg/kg po) or active congener phenyl saligenin phosphate (PSP, 2.5 mg/kg im) with and without a calcium channel blocker which ameliorated clinical signs of organophosphate-induced delayed neuropathy (nifedipine 1 mg/kg/day x 5). Calcium channel blocker administration was initiated 1 day prior to administration of organophosphate (OP). OP administration caused an increase in CANP activity in brain within 4 days and in sciatic nerve and gastrocnemius muscle within 2 days of administration. This increase did not occur if nifedipine was administered to PSP-treated hens. Total sciatic nerve calcium concentrations were also increased by PSP, but not until OP-treated hens were no longer being administered calcium blockers. This indicates that calcium channel blockers may contribute to amelioration of organophosphate-induced delayed neuropathy by attenuation of calcium-mediated disruption of axonal and muscle cytoskeletal homeostasis.

Measurement of low levels of x-ray mutagenesis in relation to human disease.

We previously demonstrated that conventional methods for measurement of mutagenesis in mammalian cells are subject to serious error that causes underestimation of environmental contributions to cancer and genetic disease. This error has been corrected by use of somatic cell hybrids containing a single human chromosome on which the marker genes are carried and by using doses of mutagenic agents so low that little cell killing occurs. This method permits direct measurement of the effects of low doses of radiation and other mutagens without resort to the controversial extrapolation procedure customarily used to estimate effects of doses in the neighborhood of actual human exposures. The new data demonstrate that the true mutagenesis efficiency at the low doses of ionizing radiation that approximate human exposures is more than 200 times greater than those obtained with conventional methods. This methodology also permits evaluation of localized mutations, large and small chromosomal deletions, and nondisjunctional processes and can be used for mutagens that need metabolic activation as well as for cooperatively acting agents. The two opposing classical views that in mammalian cells extrapolation to low doses of x-radiation is linear, on the one hand, or involves a threshold, on the other, are both demonstrated to be incorrect at least for the conditions here considered. The actual curve exhibits a downward concavity so that the mutational efficiency is maximal at low doses. These data may have important implications for human health.