Interaction of Soman with {beta}-Cyclodextrin

Interaction of Soman with ß-CycIodextrin. DESIRE,B., AND SAINT-ANDRE, S. (1986). Fun-dam. Appl. Toxicol. 7, 646-657.Of the following neurotoxic agents, pinacolyl methylphospho-nofluoridate(soman), isopropyl methylphosphonofluoridate (sarin) and ethylN, N-dimethyl-phosphoramidocyanidate (tabun), only soman wasinactivated appreciably at pH 7.40 by ß-cyclodextrin.The interaction of soman, a mixture of four stereoisomers designatedas C(+)P(–), C(–)P(–), C(+)P(+), and C(–)P(+),with cyclodextrins was revealed by methods based on (i) theirreversible inhibition of acetylcholinesterase (AChE) thatis phosphonylated chiefly by P(–)-isomers of racemic somanand (ii) continuous titration of fluoride ions released by somanusing a fluoride-specific electrode. Soman and ß-cyclodextrinform a 1:1 complex. At pH 7.40 and 25°C the dissociationconstant Kd of this complex and the rate constant k2 of cleavageof soman by ß-cyclodextrin are (0.53 ± 0.05)mM and (5.9 ± 0.6) x 10² min¹, respectively. The rateconstant k₂^max for the cleavage of soman by monoionized ß-cyclodextrinhas a value of 2.8 x 103 min¹ and the second order rate constantk₂^max/K_d 5.3 x 106 M^–1 min^–1. Consequently, somanis hydrolyzed about 2500 times faster by the monoanion of ß-cyclodextrin,than by the hydroxide ion. The cleavage of P(–)-somanby ß-cyclodextrin as estimated by AChE inhibitionproceeds apparently at the same rate for the C(–)P(–)-and C(+)P(–)-isomers. However, the release of fluorideions indicated a stereospecific rate of reaction, the P(-Hsomersreacting faster than the P(+)-isomers. At pH 7.40, the inactivationrate of soman by ß-cyclodextrin was as fast in humanplasma in vitro as in Tris buffer. This interaction betweensoman and ß-cyclodextrin, and other data from theliterature, suggests that the introduction of catalytic or noncatalyticgroups on ß-cyclodextrin might possibly make it abetter catalyst for soman inactivation through improvement inthe catalytic or in the binding process.     

Action of Organophosphates on GABAA Receptor and Voltage-Dependent Chloride Channels

The effects of several organophosphates were studied on thebinding of t-[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS)to rat brain GABA_A receptor and receptor function as assayedby GABA-induced ³⁶Cl^– influx into membrane vesicles andon the binding of [³⁵S]TBPS to a voltage-dependent Cl^–channel in Torpedo californica electric organ. The organophosphateanticholinesterases diisopropylphosphorofluoridate, soman, sarin,tabun, and VX had little or no effect on GABA-regulated chloridechannels. They also had no effect on [³⁵S]TBPS binding to thevoltage-dependent chloride channel, except for soman which inhibitedit with an IC50 of 24 µM. Triphenyl phosphate was theonly one of three organophosphate flame retardants tested thatinhibited both GABA-regulated chloride channel and binding of[³⁵S]TBPS to the voltage-dependent chloride channel with IC50sof 18 and 13 µM, respectively. The industrial organophosphatetri-o-cresyl phosphate and the anticholinesterase organophosphateinsecticides leptophos, leptophos oxon, and O-ethyl O-4-nitrophenylphenylphosphonothioate inhibited GABA-regulated chloride channelsand bound with high affinity to the voltage-dependent chloridechannels (lC50 = 0.3 to 8.7 µM). There was no apparentcorrelation between the affinities of the GABA_A receptor chloridechannel or the voltage-dependent chloride channel for the differentorganophosphates and their potencies in inhibiting acetylcholinesteraseor in inducing delayed neurotoxicity. Nevertheless, althoughthe voltage-dependent chloride channel and/or GABA_A receptorare not primary targets for organophosphate anticholinesterasesand flame retardants, it is suggested that the inhibition ofthese two proteins by certain organophosphates may contributeto their toxicities.     

The Effects of Select Neurotoxic Chemicals on Synaptosomal Monoamine Uptake and K+-Dependent Phosphatase

The Effects of Select Neurotoxic Chemicals on Synaptosomal MonoamineUptake and K⁺-Dependent Phosphatase. Bracken, William M., Sharma,Raghubir P. and Kleinschuster, Stephen J. (1981). Fundam. Appl.Toxicol. 1:432–436. The in vitro inhibition of norepinephrine(NE) and serotonin (5-HT) uptake into rat brain synaptosomesby a diverse group of neurotoxic chemicals was studied. Thetest chemicals included CH₃HgCl, Hg(NO₃)₂, CdCl₂, diisopropylfluorophosphate(DFP), paraoxon, acrylamide and Kepone while chlorpromazineand ouabain were used as reference chemicals. Methylmercuricchloride, Hg(NO₃)₂ and Kepone inhibited the NE and 5-HT uptakewith IC₅₀'s (concentration of chemical inhibiting 50% of uptake)between 10^–4 to 10^–3 M for both amines. Maximalinhibition was 60–100% at 10 3 M. Cadmium chloride, paraoxon,DFP and acrylamide were not inhibitory. The influence of thetest chemicals on synaptosomal K⁺-dependent phosphatase wasstudied. Methylmercuric chloride, Hg(NO₃)₂, CdCl₂ and Keponewere inhibitors of the phosphatase with 50% inhibition (I₅₀)at micromolar concentrations. The phosphatase was most sensitiveto Hg(NO₃)₂ inhibition with an I₅₀ of 0.03 /M. The inhibitoryconcentrations for these chemicals ranged from 10^–7 to10^–3 M. A correlation of the phosphatase and monoamineuptake inhibitions was not suggested from the data. The lowaffinity inhibition (IC₅₀ greater than 10(^–5 M) of theNE and 5-HT uptake by CH₃HgCl, Hg(NO₃)₂ and Kepone suggestedthat this is not a biologically important phenomena. The apparenthigh affinity inhibition (I₅₀ less than 10^–5 M) of thephosphatase demonstrated the specific influences the test compoundscan have on enzymatic processes. Such enzymatic inhibition wouldbe of critical importance if these neurotoxicants were ableto penetrate the synaptic or neuronal membrane.     

In Vitro 2,3,7,8-Tetrachlorodibenzo-p-dioxin Interference with the Anterior Pituitary Hormone Adrenocorticortropin

Treatment of male Sprague-Dawley rats with a single oral doseof 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shownto increase serum adrenocorticotropin (ACTH) and decrease serumcorticosterone. The present in vitro study was designed to assesswhether TCDD has a direct effect on the anterior pituitary underbasal and stimulated conditions. Primary anterior pituitarycell cultures were prepared from normal 180- to 220-g male Sprague-Dawleyrats and the cultures treated with 10^–9–10^–19M TCDD. Maximal secretion of ACTH occurred between 10^–11and 10^–15 M TCDD for both medium (2-fold) and intracellular(1.5-fold) concentrations after 24 h TCDD exposure. TCDD treatmentalso caused an early (6 h) and persistent (10 days) increasein basal medium (1.4- to 2.8-fold) and intracellular (1.1- to1.7-fold) ACTH concentrations. However, while stimulation withcorticotropin-releasing hormone (CRH) increased intracellularACTH 1.5- to 1.7-fold in pituitary cells treated for 24 h with10^–9–10^–13 M TCDD, ACTH secreted into themedia was decreased by 30–50% compared with controls.Lastly, the secretagogue arginine-8-vaso-pressin (AVP), didnot increase the amount of ACTH secreted above levels observedwith basal TCDD exposure. From this study, it appears that TCDDstimulates in vitro synthesis and secretion of ACTH by the anteriorpituitary under basal conditions, but decreases the pituitary'sresponsiveness to CRH and AVP stimulation.     

Toxaphene Inhibition of Calmodulin-Dependent Calcium ATPase Activity in Rat Brain Synaptosomes

Toxaphene Inhibition of Calmodulin-Dependent Calcium ATPaseActivity in Rat Brain Synaptosomes. PRASADA RAO, K. S., TROTTMAN,C. H., MORROW, W., AND DESAIAH, D. (1986) Fundam. Appl. Toxicol.6, 648–653. Effect of toxaphene on Ca⁺²-ATPase activityin rat brain synaptosomes was studied in vitro and in vivo.Ca⁺²-ATPase in calmodulin-depleted synaptosomes was inhibitedin vitro to a maximum of about 50% at 150 µM toxaphenc.Substrate activation kinetics of Ca⁺²-ATPase in synaptosomesrevealed that toxaphene inhibited the enzyme activity noncompetetivelyby decreasing V_max values, without affecting the enzyme-substrateaffinity. Toxaphene inhibited the calmodulin activated Ca⁺²-ATPaseactivity in a concentration-dependent manner with an IC50 of10 µM, a concentration at which no significant effectwas observed on basal enzyme activity. Nuclear and P₂ fraction(synaptosomes) calmodulin levels were reduced significantlyin toxaphene-treated rats. The synaptosomal Ca⁺²-ATPase wasalso reduced to about 45% in toxaphene-treated rats and theactivity was restored to normal levels by the exogenously addedcalmodulin. These results suggest that toxaphene may cause synapticdysfunction by in terfering with calmodulin and its regulationof neuronal calcium.     

Changes in Sertoli Cell Function in vitro Induced by Nitrobenzene

Changes in Sertoli Cell Function in vitro Induced by Nitrobenzene.ALLENBY, G., SHARPE, R. M., AND FOSTER, P. M. D. (1990). Fundam.Appl. Toxicol 14, 364–375. Nitrobenzene (NB) has beenidentified as a testicular toxicant In vivo, but its site ofaction remains unknown. In the present study, the effect ofNB on the Sertoli cell was assessed in vitro using Sertoli celland Sertoli-germ cell cocultures. The parameters measured werethe exfoliation of germ cells; the secretion of lactate, pyruvate,and inhibin; and gross cellular morphology. The effect of meta-dinitrobenzene(mDNB), a related compound which is a known Sertoli cell toxicant,was assessed for comparison. Gross morphological changes includingvacuolation of Sertoli cells were observed following treatmentof cultures with 10^–3 M NB. Exposure of cocultures toNB also resulted in dose-dependent exfoliation of predominantlyviable germ cells. NB (>5 ? 10^–4 m) and mDNB at thesingle dose level used (10^–3 m) stimulated the secretionof lactate and pyruvate significantly by Sertoli cells, an effectthat was more marked in the absence of germ cells. Comparablechanges were observed in follicle stimulating hormone (FSH)-stimulatedcultures. Inhibin secretion by Sertoli cells was also alteredby exposure to NB but in a biphasic manner, with low (10^–8to 10^–6 m) and high (10^–4 to 10^–3 m) dosesenhancing inhibin secretion while intermediate (10^–5 M)doses had no effect. These effects were evident in both culturesystems but inhibin secretion by Sertoli-germ cell cocultureswas always greater than that by Sertoli cell cultures. However,these effects of NB on inhibin secretion were not evident inFSH-stimulated cultures. In contrast to the effects of NB, mDNBhad no effect on basal secretion of inhibin but blocked thestimulatory effect of FSH. It is concluded that NB, like mDNB,is probably a Sertoli cell toxicant in view of its similar disruptiveeffects on various parameters of Sertoli cell function. However,NB is far less toxic than mDNB at equivalent concentrationsin vitro The present study is the first to evaluate the potentialof inhibin secretion by Sertoli cells in culture as an additionalmarker of toxicant action, and concludes that it merits furtherstudy in this context.     

Alterations in Particle Accumulation and Clearance in Lungs of Rats Chronically Exposed to Diesel Exhaust

Alterations in Particle Accumulation and Clearance in Lungsof Rats Chronically Exposed to Diesel Exhaust. WOLFF, R. K.,HENDERSON, R. F., SNIPES, M. B., GRIFFITH, W. C., MAUDERLY,J. L., CUDDIHY, R. G., AND MCCLELLAN, R. 0. Fundam Appl. Toxicol9, 154–166. F344 rats were chronically exposed to dieselexhaust at target soot concentrations of 0 (control, C), 0.35(low, L), 3.5 (medium, M), and 7.0 (high, H) mg/m³ Accumulatedlung burdens of diesel soot were measured after 6, 12, 18, and24 months of exposure. Parallel measurements of particle depositionand clearance were made to provide insight into the mechanismsof particle accumulation in lungs. The fractional depositionof inhaled ⁶⁷Ga₂O₃ particles after 6, 12, 18, and 24 monthsof exposure and of inhaled ¹³⁴Cs-fused aluminosilicate particlesafter 24 months were similar for all groups. Progressive increasesin lung burdens of soot particles were observed in M and H exposedrats, reaching levels of 11.5 ± 0.5 and 20.5 ±0.8 mg/lung (SE), respectively, after 24 months. Rats in theL group had smaller relative increases in lung burden, reachinglevels of 0.60 ± 0.02 mg/lung after 24 months. Trachealmucociliary clearance measurements, using ^99mTc-macroaggitatedalbumin deposited in the trachea, showed no changes at anytime.There were statistically significant increases inclearance half-timesof inhaled radiolabeled particles of ⁶⁷Ga₂O₃ as early as 6 monthsat the H level and 18 months at the M level; no significantchanges were seen at the L level. Rats inhaled fused aluminosilicateparticles labeled with ¹³⁴Cs after 24 months of diesel exhaustexposure to measure long-term components of pulmonary clearance.The long-term clearance half-times were 79 ± 5, 81 ±5, 264 ± 50, and 240 ± 50 days (± SE) forthe C, L, M, and H groups, respectively. Differences were significantbetween the C and both the M and H exposure groups (p <0.01).Lung burdens of diesel soot were more than expected at the Hand M levels and were also associated with impaired particleclearance while smaller responses were observed in both burdensand clearance at the L level.     

Effects of Chlorpyrifos on High-Affinity Choline Uptake and [3H]Hemicholinium-3 Binding in Rat Brain

High, subcutaneous doses of the organophosphorus insecticidechlorpyrifos (CPF) in adult male rats can be well-tolerateddespite extensive and persistent acetylcholinesterase (AChE)inhibition. We propose that changes in acetylcholine synthesiscould modulate the toxicity associated with extensive AChE inhibitionfollowing CPF exposure. High-affinity choline uptake (HACU,the rate-limiting step in acetylcholine synthesis) and bindingto [³H]-hemicholinium-3 (HC-3, a specific ligand for the cholinetransporter) were chosen as indicators of acetylcholine synthesis.Female, Sprague-Dawley rats (220–280 g) were treated witheither vehicle (peanut oil, 2 ml/kg, sc) or CPF (280 mg/kg,2 ml/kg, sc), examined daily for clinical signs of toxicity,and sacrificed 1, 2, or 7 days later for neurochemical measurements{AChE inhibition, muscarinic receptor binding using [³H]quinuclidinylbenzilate (QNB) and [³H]cis-methyldioxolane (CD) as ligands,HACU and [³H]HC-3 binding} in frontal cortex. Despite extensiveAChE inhibition (90–93%) at all time points, relativelyminor degrees of overt toxicity were noted in CPF-treated rats.Binding to the non-selective muscarinic antagonist [³H]QNB wasreduced (10–34%), whereas binding to the putative m2-selectiveagonist [³H]CD was increased (15–23%) at all three timepoints. HACU was reduced (20%) in crude synaptosomes preparedfrom CPF-treated rats 1 day following exposure but no significantchanges were noted at 2 or 7 days after treatment. CPF-oxon,the active oxidative metabolite of CPF, was a weak inhibitorof HACU in vitro (IC₅₀>200 µM). Binding to [³H]HC-3was reduced in a dose-related manner 1 day after CPF exposure.Kinetic analyses of [³H]HC-3 binding 1 day after CPF (280 mg/kg)indicated a significant reduction in density {B_max: control,187±18 fmol/mg protein; CPF, 104±12 fmol/mg protein)with no apparent change in binding affinity (K_d: control, 25±3nM; CPF, 19±3 nM). These results suggest that a reductionin HACU/acetylcholine synthesis may contribute, along with compensatorychanges in cholinergic receptors, to the diminished toxicityfollowing extensive AChE inhibition by CPF.     

Differences in the Toxicity of Soman in Various Strains of Mice

Differences in Toxicity of Soman in Various Strains of Mice.Clement, J.G., Hand, B.T. and Shiloff, J.D. (1981). Fundam.Appl. Toxicol. 1:419–420. The acute toxicity of somanwas assessed in eight strains of mice (ALAS, CD® -1, C57BL,CF1®, CFW® C3H, DBA and BALB/c). In fasted animals theLD₅₀ values for soman varied from 98 µg/kg in C57BL miceto 151 µg/kg in BALB/c mice. In general in non-fastedmice the soman LD⁵⁰ was not significantly changed except inALAS strain where the soman LD⁵⁰ value increased significantly.The different sensitivities to soman poisoning among the variousstrains does not appear to be due totally to differences inlevel of brain acetylcholinesterase. Fasting had no significanteffect on the activity of brain acetylcholinesterase and somantoxicity in CD® -1 mice whereas, upon fasting ALAS strainmice for 18 hr, there was a 25% decrease in brain acetylcholinesterasewhich could explain their increased sensitivity to soman however,it is possible that other biochemical changes may also playa role.     

Efficacy of Mono- and Bis-Pyridinium Oximes Versus Soman, Sarin and Tabun Poisoning in Mice

Efficacy of Mono- and Bis-Pyridinium Oximes Versus Soman, Sarinand Tabun Poisoning in Mice. Clement, J.G. (1983). Fundam. Appl.Toxicol. 3:533–535. Various oximes (PAM, toxogonin, TMB-4,HS-6, HI-6, HGG-12, HGG-42) combined with atropine were comparedas antidotes of soman, sarin and tabun poisoning in non-fastedCD-1^® male mice. TMB-4 was the most toxic oxime with ani.p. LD₅₀ value of 80 mg/kg and HI-6 was the least toxic oximewith an i.p. LD₅₀ of 588 mg/kg. Upon comparing ED₅₀ values,HGG-42 was the most effective oxime versus soman and tabun poisoningwhereas, HI-6 was the most effective oxime versus sarin poisoning.Further research needs to be done to explain the distinct differencesin efficacy of the oximes versus poisoning by soman, sarin ortabun.     

Screening of Potentially Hormonally Active Chemicals Using Bioluminescent Yeast Bioreporters

Saccharomyces cerevisiae bioluminescent bioreporter assays weredeveloped previously to assess a chemical's estrogenic or androgenicdisrupting potential. S. cerevisiae BLYES, S. cerevisiae BLYAS,S. cerevisiae BLYR, were used to assess their reproducibilityand utility in screening 68, 69, and 71 chemicals for estrogenic,androgenic, and toxic effects, respectively. EC₅₀ values were6.3 ± 2.4 x 10^–10M (n = 18) and 1.1 ± 0.5x 10^–8M (n = 13) for BLYES and BLYAS, using 17β-estradioland 5-dihydrotestosterone over concentration ranges of 2.5 x10^–12 through 1.0 x 10^–6M, respectively. Based onanalysis of replicate standard curves and comparison to backgroundcontrols, a set of quantitative rules have been formulated tointerpret data and determine if a chemical is potentially hormonallyactive, toxic, both, or neither. The results demonstrated thatthese assays are applicable for Tier I chemical screening inEnvironmental Protection Agency's Endocrine Disruptor Screeningand Testing Program as well as for monitoring endocrine-disruptingactivity of unknown chemicals in water.     

Allosteric Binding of Nickel(II) to Calmodulin

The binding of Ni(II) to calmodulin (CAM) in the presence andin the absence of Ca(II) was investigated by equilibrium dialysisin order to test the physicochemistry of direct Ni(II)-CAM interactionsthat might be responsible for the effects of this metal on CAMobserved in vivo. Samples containing 5 µm CAM, 5 mM Tris/HClbuffer (pH 7.4), and NaCl to maintain the ionic strength I =3600 µm, with or without 200 µm CaCl₂, were dialyzedat 37?C against 1–300 µm ⁶³NiCl₂. In the presenceof Ca(II), the CAM molecule has two binding sites for Ni(II)(K₁, = 7.25 ? 10⁵m^–1; = 3.79 ? 10³ M^–1) with markedcoopera-tivity (Hill coefficient = 1.20 ? 0.03 SE). In the absenceof Ca(II), a complicated Ni(II)-binding curve is obtained indicatingformation of many mutually interacting complex species. Bindingof Ni(II) to CAM in the presence of Ca(II) is inhibited slightlyby added MnCl₂ (50 µM) and very strongly by CuCl₂ andZnCl₂ (10 µm). To elucidate the mechanism of this inhibition,binding of Zn(II) (0.5–50 µm ⁶⁵ZnCl₂) to CAM inthe presence of Ca(II) (200 µM) was also studied. Themaximum molecular ratio of Zn(II) to CAM in the Zn(II)/Ca(II)/CAMcomplex approached 0.5. Thus, the observed inhibition by Zn(II)of the Ni(II) binding to Ca(II)/CAM does not involve competitionfor the same binding sites but is rather caused by a conformationalarrangement of CAM in its Ca(II)/Zn(II) complex that is differentthan the Ca(II) complex. This fact, as well as the observeddifference in binding of Ni(II) in the presence and absenceof Ca(II), stress the importance of conformation of the CAMmolecule to Ni(II) binding.     

In Vivo Percutaneous Absorption of Boric Acid, Borax, and Disodium Octaborate Tetrahydrate in Humans Compared to in Vitro Absorption in Human Skin from Infinite and Finite Doses

Literature from the first half of this century report concernfor toxicity from topical use of boric acid, but assessmentof percutaneous absorption has been impaired by lack of analyticalsensitivity. Analytical methods in this study included inductivelycoupled plasma-mass spectrometry which now allows quantitationof percutaneous absorption of ¹⁰B in ¹⁰B-enriched boric acid,borax, and disodium octaborate tetrahydrate (DOT) in biologicalmatrices. This made it possible, in the presence of comparativelylarge natural dietary boron intakes for the in vivo segmentof this study, to quantify the boron passing through skin. Humanvolunteers were dosed with ¹⁰B-enriched boric acid, 5.0%, borax,5.0%, or disodium octaborate tetrahydrate, 10%, in aqueous solutions.Urinalysis, for boron and changes in boron isotope ratios, wasused to measure absorption. Boric acid in vivo percutaneousabsorption was 0.226 (SD = 0.125) mean percentage dose, withflux and permeability constant (K_p) calculated at 0.009 µg/cm²/hand 1.9 x 10^–7 cm/h, respectively. Borax absorption was0.210 (SD = 0.194) mean percentage of dose, with flux; and K_pcalculated at 0.009 µg/cm²/h and 1.8 x 10^–7 cm/h,respectively. DOT absorption was 0.122 (SD = 0.108) mean percentage,with flux and K_p calculated at 0.01 µg/cm²/h and 1.0 x10^–7 cm/h, respectively. Pretreatment with the potentialskin irritant 2% sodium lauryl sulfate had no effect on boronskin absorption. In vitro human skin percentage of doses ofboric acid absorbed were 1.2 for a 0.05% solution, 0.28 fora 0.5% solution, and 0.70 for a 5.0% solution. These absorptionamounts translated into flux values of, respectively, 0.25,0.58, and 14.58     

In Vivo and in Vitro Percutaneous Absorption and Skin Decontamination of Arsenic from Water and Soil

The objective was to determine the percutaneous absorption ofarsenic-73 as H₃A_sO₄ from water and soil. Soil (Yolo County65-California-57-8) was passed through 10-, 20-, and 48-meshsieves. Soil retained by 80 mesh was mixed with radioactivearsenic-73 at a low (trace) level of 0.0004 µg/cm² (microgramsarsenic per square centimeter skin surface area) and a higherdose of 0.6 µg/cm². Water solutions of arsenic-73 at alow (trace) level of 0.000024 µg/cm² and a higher doseof 2.1 µg/cm² were prepared for comparative analysis.In vivo in Rhesus monkey a total of 80.1 ± 6.7% (SD)intravenous arsenic-73 dose was recovered in urine over 7 days;the majority of the dose was excreted in the first day. Withtopical administration for 24 hr, absorption of the low dosefrom water was 6.4 ± 3.9% and 2.0 ± 1.2% fromthe high dose. In vitro percutaneous absorption of the low dosefrom water with human skin resulted in 24-hr receptor fluid(phosphate-buffered saline) accumulation of 0.93 ± 1.1%dose and skin concentration (after washing) of 0.98 ±0.96%. Combining receptor fluid accumulation and skin concentrationgave a combined amount of 1.9%, a value less than that in vivo(6.4%) in the Rhesus monkey. From soil, receptor fluid accumulationwas 0.43 ± 0.54% and skin concentration was 0.33 ±0.25%. Combining receptor fluid plus skin concentrations gavean absorption value of 0.8%, an amount less than that with invivo absorption (4.5%) in the Rhesus. These absorption valuesdid not match current EPA default assumptions. Washing withsoap and water readily removed residual skin surface arsenic,both in vitro and in vivo. The partition coefficient of arsenicin water to powdered human stratum corneum was 1.1 x 10⁴andfrom water to soil it was 2.5 x 10⁴. This relative similarityin arsenic binding to powdered human stratum corneum and soilmay indicate why arsenic absorption was similar from water andsoil. This powdered human stratum corneum partition coefficientmodel may provide a facile method for such predictions.     

Evaluation of Changes in the Secretion of Immunoactive Inhibin by Adult Rat Seminiferous Tubules in Vitro as an Indicator of Early Toxicant Action on Spermatogenesis

Evaluation of Changes in the Secretion of Immunoactive Inhibinby Adult Rat Seminiferous Tubules in Vitro as an Indicator ofEarly Toxicant Action on Spermatogenesis. Allenby, G., Foster,P. M. D., and Sharpe, R. M. (1991). Fundam. Appl. Toxicol 16,710–724. A method for culturing isolated seminiferoustubules (ST) from adult rats for 1–3 days has been developedand optimized rigorously on the basis of the secretion of immunoactiveinhibin under basal conditions and after maximal stimulationwith rat FSH or dibutyryl cyclic AMP. The effect on these culturesof three known testicular toxicants was assessed. Of these,two are thought to act on the Sertoli cell, meta-dinitrobenzene(mDNB) and nitrobenzene (NB), while the third, methoxy aceticacid (MAA), is thought to act on pachytene spermatocytes. Inaddition, the effect of a possible testicular toxicant, 3-mononitrotoluene(3-MNT), was investigated. These data were compared with thoseobtained using cultures of immature rat Sertoli cells (SC) orSC + germ cells and with data on the effect of equivalent dosesof the compounds on the secretion of immunoactive inhibin invivo. In studies designed to optimize conditions for the secretionof immunoactive inhibin by ST in culture, significant effectswere found of the type of culture medium used, the durationof culture, the total and individual length of tubules used,etc. All subsequent studies with toxicants utilized optimalconditions. Addition of either mDNB or NB to ST cultures at10^–5 or 10^–3 m, or MAA at 10^–4 m, stimulatedbasal secretion of immunoactive inhibin by two- to fourfoldon Days 1, 2, or 3 of culture while FSH or dibutyryl cyclicAMP-stimulated secretion of immunoactive inhibin was eitherunaffected or was enhanced to a small extent. At the same doses,mDNB or NB also enhanced secretion of immunoactive inhibin bySC cultures, although these effects were more variable and ofsmaller magnitude than the effects on ST cultures. In contrast,addition of up to 10^–3 m MAA to cocultures of SC + germcells had no effect on the secretion of immunoactive inhibin.Exposure of rats in vivo to levels of mDNB, NB, or MAA similarto those which stimulated secretion of immunoactive inhibinin vitro resulted in a two-to fourfold increase in the levelsof immunoactive inhibin in testicular interstitial fluid (IF)at 1 and 3 days post-treatment, and this was associated withearly impairment of spermatogenesis (as judged by testis weight).In contrast to these effects, addition of 3-MNT to ST or SCcultures had no effect except at 10^–3 m, when the secretionof immunoactive inhibin was increased marginally. Treatmentof rats with an equivalent dose of 3-MNT in vivo resulted indeath, but exposure to the highest nonlethal dose (1 g/kg) hadno significant effect either on spermatogenesis or on the levelsof immunoactive inhibin in testicular IF. In view of these findings,it is concluded that modulation of the secretion of immunoactiveinhibin by isolated ST from adult rats has considerable potentialas an in vitro screening method for investigating potentialadverse effects of chemicals on spermatogenesis, and thus meritsmore detailed evaluation. Moreover, because of the agreementbetween in vitro and in vivo findings, measurement of the levelsof immunoactive inhibin levels in vivo in testicular IF (orblood) may also be useful in the detection of early adverseeffects of chemicals on spermatogenesis.     

Carbofuran Metabolism and Toxicity in the Rat

Carbofuran Metabolism and Toxicity in the Rat. FERGUSON, P.W., DEY, M. S., JEWELL, S. A., AND KRIEGER, R. I. (1984). Fundam.Appl. Toxicol. 4, 14–21. The influence of carbofuran metabolismon acetylcholinesterase inhibition has been defined after lowdose (50 µg/kg, iv and oral) [carbonyl-¹⁴C]carbofuranexposures to male Sprague–Dawley Rats. Red blood cellacetylcholinesterase (RBC AchE) inhibition (83% at 2 min, 37%at 15 min for iv and oral, respectively, with recovery by 3hr), was correlated with carbofuran plasma concentrations (r= 0.97). Eight-hour sample collection indicated that ultimatecarbofuran fate (41–47% ^l4CO₂, 14–15% urine, <1%feces, and 30–31% carcass) was independent of exposureroute. Carbofuran absorption (peak plasma levels < 7 min),distribution, and elimination (t? = 29 ? 5 min) occurred rapidly.3-Hydroxycarbofuran, a significant oxidative metabolite of carbofuranwith anticholinesterase activity, was rapidly formed and subjectto enterohepatic circulation (plasma t? = 64 ? 5 min). Resultsindicated that rapid RBC AchE recovery closely paralleled carbofuranmetabolism and the primary in vivo disposition of 3-hydroxycarbofuranwas metabolic conjugation.     

Effect of Manganese on the Hepatic Microsomal Mixed Function Oxidase Enzyme System in the Rat

Effect of Manganese on the Hepatic Mixed Function Oxidase EnzymeSystem in the Rat , M. J., and SCHNELL, R. C. (1984). Fundam.Appl. Toxicol. 4, 1009–1018. Experiments were conductedto examine the effect of manganese on the hepatic mixed functionoxidase system in the rat Acute treatment with manganese chloride(1–10 mg Mn/kg, ip) produced a significant prolongationof hexobarbital hypnosis in male rats on Days 2 and 3 followingmetal administration. The threshold dose of manganese to producethis alteration in response was 5 mg Mn/kg and the altered responsereturned to control values by Day 5. The prolonged hexobarbitalhypnosis resulted from Mn inhibition of the hepatic microsomalmixed function oxidase system, the activity of which was assessedusing aniline (23%), ethylmorphine (26%), and hexobarbital (27%)as substrates. Manganese treatment also produced significantlyreduced levels of cytochrome P-450 (23%) and b₅ (21%), but thesubstrate-induced spectral binding of all three substrates wasnot altered significantly by Mn when expressed as A per nanomoleof cytochrome P-450. The activity of NADPH cytochrome c reductasewas also significantly decreased (25%) by Mn treatment Followingthe in vitro addition of Mn in concentrations ranging from 1x 10^–6 to 1 x 10^–3 M Mn to microsomes derived fromnaive rats, there was no decrease in the metabolism of anilineor hexobarbital or cytochrome P-450 levels. Significant inhibitionin ethylmorphine metabolism was observed with Mn concentrationsof 1 x 10^–4 m and greater. These experiments indicatethat acute Mn treatment can alter drug response as the resultof decreased hepatic biotransformation which occurs by an indirectmechanism.     

Adrenocortical Toxicity of 3-Methylsulfonyl-DDE in Mice: II. Mitochondrial Changes following Ecologically Relevant Doses

Transmission electron microscopy was used to characterize earlyultrastructural lesions in the adrenal zona fasciculata of femaleC57BL mice given a single ip injection of the adrenocorticolyticDDT-metabolite 3- methylsulfonyl-DDE (MCSO₂-DDE Following 3mg/kg, mitochondrial changes were observed 6 hr after dosing.At 12 and 24 hr the mitochondrial changes were conspicuous,with disorganization and disappearance of central cristae. Atdoses of 6, 12, and 25 mg/kg body wt initial (6 hr) mitochondrialvacuolization was observed, followed by disappearance of mitochondria(6–12 mg/kg) or cellular necrosis (25 mg/kg). The metabolicactivation and binding of MeSO₂-[¹⁴C]DDE in adrenal homogenateswere determined in vitro. The irreversible binding of MeSO₂-[¹⁴C]to the mitochondria-containing adrenal S-9 pellet fraction was50 times higher than that to the postmitochondrial S-12 supernatantfraction. The apparent K_m was 2.1 µM and the apparentV_max was 104 pmol/mg protein/30 mm for the binding of MeSO₂-[¹⁴C]to S-0.3 supernatants. The irreversible protein binding wasinhibited by metyrapone (K₁=1 µM) and 11-deoxycorticosterone(K₁=3 µM). In conclusion, the adrenal metabolic activationof MeSO₂-[¹⁴C]DDE is suggested to be mediated by a mitochondrialcytochrome P450 form, presumably P450 (11ß). A primarymitochondrial lesion develops and subsequently leads to degenerationand necrosis of the zona fasciculata.     

Pulmonary Effects to Repeated Exposures to Paraquat Aerosol in Guinea Pigs

Pulmonary Effects of Repeated Exposures to Paraquat Aerosolin Guinea Pigs. BURLEIGH-FLAYER, H. AND ALARIE, Y. (1988). Fundam.Appl. Toxicol 10, 717–729. Exposure to paraquat, a widelyused herbicide, has been shown to produce a concentration dependentrapid, shallow breathing pattern in guinea pigs 18 hr followingexposure (H. Burleigh-Flayer and Y. Alarie, 1987, Arch Toxicol.59(6), 391–396). To further explore the pulmonary effectsfollowing exposure to paraquat, two experiments were carriedout. The first experiment consisted of exposing a group of guineapigs for a period of 4 hr to 0.7 mg/m³ paraquat aerosol andmonitoring respiratory variables for 2 weeks following the exposure.In the second experiment, three groups of guinea pigs were repeatedlyexposed to three concentrations of paraquat aerosol (0.1,0.4,and 0.8 mg/m³) for 6 hr a day, 5 days a week for 3 weeks. Respiratoryvariables were measured each day of these 3-week experiments.The respiratory variables evaluated in both experiments weretidal volume (VT) and respiratory frequency (/). These variableswere monitored during air breathing and upon challenge with10% CO₂ in 20% O₂ and 70% N₂ in order to evaluate the pulmonaryeffects of exposure to paraquat. Following a single exposureto 0.7 mg/m³ paraquat aerosol, a decrease in VT and increasein f were seen during air and 10% CO₂ challenge which reacheda maximum several days following exposure. After reaching maximalchanges, the respiratory variables returned to control values.With repeated 6-hr exposures to paraquat aerosol, guinea pigsexposed to 0.4 and 0.8 mg/m³ also displayed a rapid, shallowbreathing pattern. Adaptation to the exposures for these twoconcentration groups was evidenced by a return of the respiratoryvariables toward control levels. This adaptation typically occurredduring the first 7 days of exposures. A cumulative effect wastherefore not detected with repeated exposures to paraquat aerosols.