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.     

Vitamin A Potentiation of Vinylidene Chloride Hepatotoxicity in Rats and Precision-Cut Rat Liver Slices

Pretreatment of large doses of vitamin A (VA) is known to potentiatethe hepatotoxicity of carbon tetrachloride. Therefore the effectsof 1-day VA pretreatment on VDC hepatotoxicity was examinedboth in vivo and in an in vitro system of precision-cut ratliver slices. Male Sprague-Dawley rats were pretreated with250,000 IU/kg VA by oral gavage. After 24 hr rats were administered50, 100, or 200 mg/kg VDC ip. Precision-cut liver slices wereprepared from VA pretreated rats 24 hr later and the liver sliceswere exposed for 2–8 hr to 0.025–1.0 µl VDCevaporated into the gas phase of the incubation vials. VA pretreatmentresulted in an enhancement of VDC toxicity, both in vivo andin vitro. There was a dose-dependent increase in plasma ALT24 hr after VDC treatment of rats and an increase in K⁺ leakagefrom liver slices after VDC exposure. Histological analysisof the liver or the liver slices revealed that VA+VDC treatmentresulted in centrilobular necrosis of the liver. When GdCl₃(10 mg/kg iv) was administered just before VA pretreatment ofrats, VDC toxicity was partially reversed as observed by a decreasein ALT in vivo and a decrease in the loss of K⁺ in vitro. Theseresults indicated that Kupffer cells, the resident macrophagesof the liver, were partially responsible for the VA-potentiatedVDC hepatotoxicity. One-day pretreatment of VA induced cytochromeP450IIE1 protein content as well as its enzymatic activity asmeasured by p-nitrophenol hydroxylation. Because VDC is bioactivatedby cytochrome P450IIE1, the increase in VDC hepatotoxicity afterVA may be due to an increased bioactivation of VDC in the liverand in precision-cut liver slices. Thus, more than one mechanismmay be involved in the VA enhancement of VDC hepatotoxicity.     

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.     

Acute, Distribution, and Subchronic Toxicological Studies of Succinate Tartrates

The estimated single-dose oral toxicity (50% lethality) of succinatetartrates (ST) was 2–3 g/kg in rats. ST produced minimalto moderate dermal irritation but no evidence of systemic toxicityin a standard acute percutaneous toxicity test in rabbits. STwas not an eye irritant in a standard rabbit low-volume eyeirritation test ST was not genotoxic in a series of six genotoxicitytests. A 14-day oral gavage study in rats at a dose range of0.05–1.0 g ST/kg/day produced only gastric irritation.The no-observed-effect level (NOEL) for gastric irritation was0.1 g/kg for males and 0.05 g/kg for females. A 28-day percutaneoustoxicity study in rabbits produced minimal to moderate dermalirritation and no adverse systemic effects at a high dose of450 mg ST/kg/day. Single-dose absorption, distribution, andelimination (ADE) studies in male rats showed that 10–15%of an oral dose and 1–3% of a dermal dose were absorbed.Approximately 98% of the orally administered ST was eliminatedas ¹⁴C in urine, feces, or expired CO₂ after 72 hr. Approximately80% of the dermally absorbed ¹⁴C dose was eliminated in urine,feces, or expired CO₂ after 72 hr. In conclusion, no adverseeffects were noted in acute toxicity, genotoxicity, or subchronictoxicity studies conducted with ST.     

The Distribution, Elimination, and in Vivo Biotransformation of Aldicarb in the Rainbow Trout (Oncorhynchus mykiss)

The Distribution, Elimination, and in Vivo Biotransformationof Aldicarb in the Rainbow Trout (Oncorhynchus mykiss). SCHLENK,D., ERICKSON, D. A., LECH, J. J., AND BUHLER, D. R. (1992).Fundam. Appl. Toxicol. 18, 131–136. The distribution and elimination of[¹⁴C]aldicarb, administeredorally and by intraperitoneal (ip) injection, was examined inthe rainbow trout (Oncorhynchus mykiss). Tissue residues weredetermined by monitoring radioactivity at various time periodsup to 96 hr in trout administered [¹⁴C]aldicarb orally. Periodicwater samples and a single tissue residue radioactivity levelwere obtained after 24 hr in free swimming and spinally transectedfish which received [¹⁴C]aldicarb via intraperitoneal injection.Aldicarb appears to be absorbed rapidly (99% within 3 hr) anddistributed to all tissues. Elimination profiles from both dosagegroups demonstrate a rapid a phase (oral 24 hr; ip 3 hr) probablydue to branchial excretion (96% after ip injection) followedby a slower ß phase (oral 107 hr; ip 28 hr) suggestinga deeper compartment such as muscle. [¹⁴C]Aldicarb and/or itsmetabolites were slowly being transported to the bile after24 hr. The in vivo biotransformation of [¹⁴C]aldicarb was examinedin spinally transected trout 24 hr after ip injection. The majormetabolite found was aldicarb sulfoxide (7.6%) along with lesseramounts of aldicarb oxime (5.4%).     

Adduction of Hemoglobin and Albumin in Vivo by Metabolites of Trichloroethylene, Trichloroacetate, and Dichloroacetate in Rats and Mice

Adducts to macromolecules from trichloroethylene formed by invivo and in vitro metabolism have been reported by many investigators.We examined the in vivo adduction of the blood proteins hemoglobin(Hb) and albumin in rats and mice dosed orally with [¹⁴C]trichloroethylene([¹⁴C]TRI) to explore the development of a protein adduct biomarkerof TRI exposure. We also examined the adduction of these twoproteins from doses of [¹⁴C]trichloroacetate (TCA) and [¹⁴C]dichloroacetate(DCA), two metabolites of TRI. Association of label with albuminpeaked at 4–8 hr in the rat (2480 nmol eq TRI/mg protein)and 2–4 hr in the mouse (1580 nmol eq TRI/mg protein).The decay was exponential with a half-life consistent with thatof rat or mouse albumin (approx 24 hr). The time course of labelwith Hb was characterized by an early plateau at 8 hr in rat(28 nmol eq TRI/ mg protein), 4 hr in mouse (7 nmol eq TRI/mgprotein), and followed by a slow steady increase, peaking at120 hr (54 nmol eq TRI/mg protein, rat; 38 nmol eq TRI/mg protein,mouse). This apparent binding was linear with dose in the rat,but was convex in the mouse albumin (mouse Hb label was belowdetection at low dose). We also found that a portion of theirreversibly associated label, referred to by previous investigatorsas "binding," could be accounted for as metabolic incorporationof label into glycine and serine. The fraction accounted forby metabolic incorporation was constant in albumin (approximately), while in Hb, this portion was time dependent, approximately30% at the early sampling time, 75% at the late time, implyingthe observed late increase could be accounted for by metabolicincorporation. TCA and DCA also formed Hb and albumin adducts.Portions of this binding was also due to metabolic incorporation.The pattern of the binding from TCA in albumin was differentfrom that of TRI, implying a route to adduct from TRI whichdoes not proceed through TCA.     

Age-Related Percutaneous Penetration of 2-sec-Butyl-4,6-dinitrophenol (Dinoseb) in Rats

[¹⁴C]Dinoseb was applied to previously clipped back skin of33- and 82-day-old female Fischer 344 rats at a dosage rangeof 210–2680 nmol/cm². Radioactivity in the treated skin,tissues, urine, and feces was determined at 1, 6, 24, 48, 72,and 120 hr following dermal application. In vitro dermal absorptionof [¹⁴C]dinoseb was also measured in rats of the same age bystatic and flow-through methods. In vivo dermal absorption inboth young and adults appeared biphasic with 55.6 and 82.7%of the recovered dose, respectively, penetrating in 72 hr. Invitro measurements of skin absorption at 72 hr with static cellsshowed higher values in young and lower values in the adultcompared to in vivo dermal absorption values, In vitro flow-throughmeasurements at 72 hr gave lower dermal absorption values forboth young and adult rats, compared to In vivo values. Followingin vivo application, adults excreted about 70% of the totalrecovered dose in urine, 16% in feces, and retained 7% in thebody at 120 hr. HPLC analysis of urine collected at 24 hr fromadults administered [¹⁴C]dinoseb showed extensive metabolismof parent. Excretion and retention results for young were about80% of the adult values, which also was the young to adult ratioof dermal penetration. Blood had the highest concentration ofdinoseb-derived radioactivity of the tissues examined. The kidneyto blood ratio averaged 0.60 in young and 0.41 in adults, whilethe liver and carcass to blood ratio averaged 0.18 in youngand 0.11 in adult. Dermal absorption in young rats was slightlyless than that in adults, and the subsequent kinetics of retentionand excretion appeared different, In vitro dermal penetrationof dinoseb was usually lower than In vivo absorption.     

Percutaneous Absorption, Metabolism, and Hemolytic Activity of n-Butoxyethanol

Percutaneous Absorption, Metabolism, and Hemolytic Activityof n-Butoxyethanol. BARTNIK, F. G., REDDY, A. K., KLECAK, G.,ZIMMERMANN, V., HOSTYNEK, J. J., and KUNSTLER, K. (1987). Fundam.Appl. Toxicol. 8, 59–70. A series of studies was conductedto examine the percutaneous absorption, distribution, excretion,and hemolytic activity of n-butoxyethanol (BE). Rats receivinga subcutaneous dose of ¹⁴C-labeled BE excreted the radioactivityin the urine (79%), expired air (10%), and feces (0.5%) within72 hr. Of the organs analyzed, thymus and spleen showed elevatedspecific radioactivities as compared with blood. A percutaneousapplication of BE on rats, under nonocclusive conditions, showed25–29% absorption within 48 hr. Peak blook levels of BEoccurred at 2 hr after application; butoxyacetic acid (BAA)was found to be the major metabolite. Comparison of in vitroskin penetration data showed the following absorption patternof BE: hairless rat >> pig > human skin. Hemolysisand associated hematological changes were noted in the ratswhich received single dermal applications of 260–500 mg/kgof BE. In vitro, BAA showed markedly greater hemolytic abilityon rat erythrocytes than did BE. Human erythrocytes showed nohemolysis when incubated with BE or BAA at concentrations thatare hemolytic to rat erythrocytes. An intravenous dose of 62.5mg/kg of BE does not result in hemolysis or hemoglobinuria inthe rat. The rat may be an animal model with increased susceptibilityto the effects of BE compared with humans because of its rapidpercutaneous absorptive ability and its greater hemolytic sensitivity.     

Assessment of the Developmental Toxicity, Metabolism, and Placental Transfer of N,N-Dimethylformamide Administered to Pregnant Rats

This study evaluates the developmental toxicity and placentaland milk transfer of N,N-dimethylformamide (DMF) in rats. Sprague-Dawleyrats were given 0, 50, 100, 200, and 300 mg DMF/kg/day, by gavage,on Gestational Days (GD) 6 through 20. Maternal toxicity wasindicated by depressions in weight gain and food consumptionat doses 100 mg/kg. Fetal toxicity was indicated by decreasedfetal body weight at doses 100 mg/kg, and by increased incidencesof two skeletal variations (absent or poorly ossified supraoccipitaland sternebrae) at 200 and 300 mg/kg. Thus, the maternal anddevelopmental no-observed-adverse-effect level was 50 mg/kg/day.The time course disposition of [¹⁴C]DMF was examined over a48-hr period in GD12- and GD18-pregnant rats after a singleoral dose of 100 mg [¹⁴C]DMF/kg Peak concentrations of radiocarbonoccurred within 1 hr after dosing. Embryonic (GD 12) and fetal(GD18) tissues accounted for 0.15 and 6% of the administereddose, respectively. Levels of radiocarbon in embryonic and fetaltissues were equal or slightly less than in maternal plasmaup to 8 and 24 hr, respectively, and higher thereafter. HPLCanalysis performed at intervals from 1 to 8 hr on GD12 and 1–24hr on GD18 indicated that unchanged DMF and metabolites werereadily transferred to the embryonic and fetal tissues, wheretheir levels were generally equal to those in maternal plasma.The parent compound accounted for most of the radioactivityuntil 4–8 hr and then decreased. N-Hydroxymethyl-N-methylformamide(HMMF) and N-methylformamide (NMF) were the predominent metabolitesand increased with time. Much lower concentrations were foundfor formamide and N-acetyl-S-(N-methylcarbamoyl)cysteine. Transferof radioactivity into milk was studied in dams given a singleoral administration of 100 mg [¹⁴C]DMF on Lactation Day 14.DMF, HMMF, and NMF were found in the milk at concentrationsequal to those in plasma.     

Glyphosate Skin Binding, Absorption, Residual Tissue Distribution, and Skin Decontamination

Glyphosate Skin Binding, Absorption, Residual Tissue Distribution,and Skin Decontamination. Wester, R. C., Melendres, J., Sarason,R., McMaster, J., and Maibach, H. I. (1991). Fundam. Appl. Toxicol.16, 725–732. Glyphosate is a broad-spectrum postemergencetranslocated herbicide. Its interactions with skin and potentialsystemic availability through percutaneous absorption was studiedby skin binding, skin absorption, residual tissue distribution,and skin decontamination. Glyphosate in a final formulation(Roundup) undiluted and diluted with water 1:20 and 1:32, wouldnot partition into powdered human stratum corneum (<1%).In vitro percutaneous absorption through human skin into humanplasma as receptor fluid was no more than 2% over a concentrationrange of 0.5–154 µg/cm² and a topical volume rangeof 0.014–0.14 ml/cm². Disposition of glyphosate followingiv administration of 93 and 9 µg doses to rhesus monkeyswas mainly through urine excretion, 95 ± 8 and 99 ±4% in 7 days, respectively. Percutaneous absorption in vivoin rhesus monkey was 0.8 ± 0.6% for the low dose (25µg/cm²) and 2.2 ± 0.8% for the high dose (270 µg/cm²).No residual ¹⁴C was found in organs of the monkeys euthanized7 days after the topical application. Washing the skin applicationsite with soap and water removed 90 ± 4% of applied dose,and washing with water only removed 84 ± 3% of applieddose. Both soap and water and water only were equal in abilityto remove glyphosate from skin over a 24 hr skin applicationperiod. About 50% of the initially applied dose could be recoveredafter 24 hr. Glyphosate is very soluble in water and insolublein most organics (octanol/water log P = –1.70) and thereforenot compatible with the lipid-laden stratum corneum. This isconsistent with the low skin binding and skin absorption andalso consistent with the efficient removal from skin with soapand water or water-only wash.     

Amelioration of Bromobenzene Hepatotoxicity in the Male Rat by Zinc

Amelioration of Bromobenzene Hepatotoxicity in the Male Ratby Zinc. MCMILLAN, D. A., AND SCHNELL, R. C. (1985). Fundam.Appl. Toxicol. 5, 297–304. Experiments were conductedto examine the role of zinc in the prevention of bromobenzenehepatoxicity in male rats. Bromobenzene (BB) (7.5 mmol/kg, ip)produced a marked hepatotoxicity as evidenced by increases inplasma alanine aminotransferase (ALT) and aspartate aminotransferse(ASP) activities and a marked depression in hepatic glutathione(GSH) content 24 hr after administration. The administrationof zinc (92 µmol Zn/kg, ip, at 48 and 24 hr prior to thebromobenzene) ameliorated the bromobenzene elevations in plasmaAST (25%) and plasma ALT (50%) but did not alter the decreasesin hepatic GSH. Following administration of [¹⁴C]BB, the radioactivelabel was distributed primarily in the cytosolic and lipid fractionsderived from liver homogenates. Furthermore, the subcellulardistribution of [¹⁴C]BB was not altered by zinc pretreatment.The extent of covalent binding of [¹⁴C]BB metabolites to hepatictissue was significantly depressed in zinc-treated rats. Zincinduced the hepatic levels of metallothionein but [¹⁴C]BB didnot bind to this sulfhydryl rich protein. Further experimentsshowed that zinc treatment depressed cytochrome P-450 content,the activity of NADPH cytochrome c reductase, and the metabolismof aniline, but not that of ethylmorphine. These studies suggestthat the hepatoprotective effect of zinc against bromobenzenetoxicity does not involve altered binding of the reactive toxicmetabolite to glutathione or metallothionein, but it may bemediated by the inhibitory effect of zinc on the microsomalcytochrome P-450-dependent drug metabolizing system.     

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.     

Biliary Excretion Appears Rate Limiting for Hepatic Elimination of Benzo[a]pyrene by Temperature-Acclimated Rainbow Trout

Biliary Excretion Appears Rate Limiting for Hepatic EliminationOf Benzo[a]pyrene by Temperature-Acclimated Rainbow Trout. CURTIS,L. R., FREDERICKSON, L. K., AND CARPENTER, H. M. (1990) Fundam.Appl. Toxicol. 15, 420–428. Previous work demonstratedthat mixed function oxidase activities of hepatic microsomesfrom cold- and warm-acclimated rainbow trout were similar whenassayed at temperatures to which fish were acclimated. This"ideal temperature compensation" was partially explained byconstitutive differences in microsomes. In the work reportedhere, rainbow trout were acclimated at 10 or 18°C for 4weeks and then ip injected with 10 µmol [³H] or [¹⁴C]benzo[a]pyrene(BP)/kg in one of two temperature regimens. First, fish wereacclimated and exposed at the same temperature and killed after4, 24, or 48 hr. Concentrations of [³H]BP equivalents in liver,bile, and fat but not in plasma, muscle, intestine, gill, orkidney increased with time. There were no differences in hexaneor ethyl acetate extract-able [³H] or [¹⁴C]BP tissue concentrationsin 10 and 18°C-acclimated fish exposed at their acclimationtemperatures. At 24 hr after injection, biliary excretion of[³H]BP equivalents was about twofold higher at 18°C thanat 10°C. Therefore, warmer temperature stimulated biliaryexcretion without a marked effect on in vivo BP metabolism.In the second regimen, 10 and 18°C-accli-matedfish wereshifted to 14°C, injected with [³H] or [¹⁴C]BP 1 hr later,and killed after an additional 24 hr. There were no differencesin tissue concentrations of total [³H]BP equivalents betweenacclimation groups at 14°C. However, the biliary concentrationof [¹⁴C]BP not extracted by ethyl acetate was significantlyhigher in bile from 10°C-acclimated fish than from 18°C-acclimatedfish when both groups were exposed at 14°C. In this case,in vivo BP metabolism was altered without coincident effectson biliary excretion. Aryl hydrocarbon hydroxylase assayed at14°C was about threefold higher in hepatic microsomes from10°C-acclimated fish than from 18°C-acclimated fish.Exposure temperature selectively modulated BP metabolism andbiliary excretion in temperature-acclimated rainbow trout.     

Metabolism of {alpha}-Olefin Sulfonate (AOS) in Rats

Metabolism of -Olefin Sulfonate (AOS) in Rats. Inoue, S., O'Grodnick,J.S. and Tomizawa, S. (1982). Fundam. Appl. Toxicol. 2:130-138.The metabolic fate of ¹⁴C-AOS (a mixture of ¹⁴C-sodium alkenyl(2)sulfonate and ¹⁴C-sodium 3-hydroxy alkane sulfonate) has beenstudied in rats by a single oral and intravenous injection of100 mg (50 µCi)/kg and 10 mg (5 µCi)/kg, respectively.After oral administration, ¹⁴C-AOS was rapidly absorbed fromthe gastrointestinal tract. The blood level of ¹⁴C-activityreached its peak 3 hr after dosing and then declined. Twenty-fourhr after the dose, about 0.8%/g of the ¹⁴C-AOS given was detectedin cecum content, but in other tissues the figures were under0.02% dose/g. Within 24 hr after the dose, 72% of the dose wasexcreted in the urine and 22% in the feces, while the excretionin the bile was 4.3% within 12 hr. The administered radioactivitywas rapidly eliminated from the whole body within 24 hr. Afterintravenous injection, half of the administered dose of radioactivitywas excreted within 1 hr. In the 0–6 hr interval post-dose,90% of the dose was excreted in the urine. No intact ¹⁴C-AOSwas detected in any of the urine samples after oral and intravenousdoses. The metabolite was apparently more polar than intact¹⁴C-AOS, and results from data of electrophoresis and equilibriumdialysis indicated that intact ¹⁴C-AOS can bind with proteins,while the metabolites cannot. The metabolite was found to containalcoholic, unsaturated and sulfonic acid functionalities. Itis suggested that the metabolite may be a hydroxylated or polyhydroxylatedsulfonic acid of shorter chain length than AOS. These resultssuggest that ¹⁴C-AOS is rapidly absorbed, metabolized and excreted,therefore, no accumulation of ¹⁴C-AOS occurs.     

Assessment of the Developmental Toxicity and Placental Transfer of 1,2-Dichloroethane in Rats

This study evaluates the developmental toxicity and placentaltransfer of 1,2-dichloroethane (DCE) in rats. Sprague–Dawleyrats were given 0–2.4 mmol DCE kg^-1 day^-1 by gavage, orwere exposed for 6 hr per day to 0–300 ppm DCE by inhalation,from Day 6 to 20 of gestation. Maternal toxicity was observedafter inhalation exposure to 300 ppm DCE and oral administrationof 2.0 or 2.4 mmol DCE kg^-1 There was no evidence of alteredgrowth nor teratogenic effects after either inhalation or oraladministration of DCE at any concentration tested. The timecourse disposition of ¹⁴C was examined over a 48-hr period in12- and 18-day pregnant rats after a single oral dose of 1.6mmol [¹⁴C]DCE kg^-1. Peak concentrations of radiocarbon occurredbetween 2 and 4 hr postdose. Conceptus (Day 12) and fetal (Day18) tissues accounted for 0.06 and 0.4% of the administereddose, respectively. Up to 4 hr, levels of radiocarbon in placentaand fetus were slightly less than in maternal plasma of 18-daypregnant rats and were two to five times higher at later periods.At 2 hr, unchanged DCE accounted for most of radioactivity (78–86%)recovered in maternal plasma, placenta, and fetus. Acidic metabolitesand radioactivity bound to macromolecules increased up to 24hr (0.01 µmol-eq DCE g^-1)in either placental or fetaltissues. Thereafter, their levels declined more slowly thanthose in the maternal plasma. Results from this developmentaltoxicity study in rats confirm embryonic exposure to radiocarbonassociated with [¹⁴C]DCE and/or its metabolites and has demonstratedthe lack of observable teratogenic effects.     

Pulmonary Immunotoxicity of Inhaled Ammonium Metavanadate in Fisher 344 Rats

Male Fisher 344 rats were exposed to 2 mg vanadium(V)/m³ (asammonium metavanadate NH⁴VO³, 0.32 µm MMD) atmospheresfor 8 hr/day for 4 days in a nose-only exposure system. In exposedrats, lung V burdens increased in a time-dependent fashion.Analysis of lung cells and lavage fluid 24 hr after the finalexposure suggested that tissue damage and a strong inflammatoryresponse was elicited; numbers of neutrophil and small macrophages(M), as well as levels of lavageable protein and lactate dehydrogenase,were significantly elevated as compared with levels observedwith air-exposed rats. Vanadium also affected pulmonary alveolarM (PAM) capacities to produce and respond to immunoregulatingcytokines. Inducible PAM production of tumor necrosis factor-awas significantly inhibited, as was the ability to increasecell surface Class II/I-A molecule expression in response tointerferon- (rFN-). PAM from V-exposed hosts were also inhibitedin their ability to be primed by EFN- to produce superorideanion and hydrogen peroxide in response to stimulation withopsonized zy-mosan. These studies indicate that short-term repeatedexposure of rats to atmospheric V, at levels encountered inan occupational setting, can alter host pulmonary immunomocompetence,with one major effect occurring at the level of cytokine-relatedfunctions. These alterations may be underlying mechanisms forthe well-documented increases in bronchopulmonary infectionsand cancers in workers chronically exposed to V-containing atmospheres.     

Chlordecone Pretreatment Alters [14C]Chlordecone and [14C]Cholesterol Transport Kinetics in the Perfused Rat Liver

Previous work demonstrated that pretreatment of mice with lowdoses of the organochlorine insecticide chlordecone (CD) alteredthe tissue disposition of a subsequent [¹⁴C]CD or [¹⁴C]- cholesterolchallenge dose. The profile of these changes was consistentwith the induction of a protein integral to hepatic CD/cholesterolturnover. The present study was undertaken to confirm similarin vivo effects in the rat and to analyze potential CD-inducedchanges in hepatic transport kinetics in the per fused rat liver.For in vivo experiments, male, Sprague-Dawley rats were treatedwith CD (5, 15, or 40 mg/kg) and challenged 3 or 7 days laterwith a 5 mg/kg [¹⁴C]CD tracer dose. Rats challenged 3 days aftertreatment and evaluated 16 hr later showed a dose-dependentdecrease in hepatic [¹⁴C]CD relative to controls. This decreasecould not be attributed to alterations in liver mass or totalliver lipid. For kinetics studies, rats received 15 mg/kg CDand livers were perfused 3 days later. Following a brief (5–7min) single-pass perfusion, the perfusate was replaced withrecirculating buffer containing albumin-bound [³H]oleic acidor high-density lipoprotein-bound [¹⁴C]CD or [¹⁴C]cholesterolLivers from pretreated animals had significantly decreased ratesof [¹⁴C]CD and [¹⁴C]cholesterol uptake. Efflux of [¹⁴C]CD andbiliary excretion of [¹⁴C] were increased. No changes were observedin uptake or biliary excretion of [³H]oleic acid. SDS-PAGE ofhepatic cytosol revealed an enhanced band in tensity correspondingto a M_r of 25,600 in livers from pretreated rats. These resultsare supportive of a competitive interaction between cholesteroland CD for proteins associated with hepa tocellular transportand excretion and suggest that CD pretreatment may have an inductiveeffect on these proteins.     

Variable Effects of Soman on Macromolecular Secretion by Ferret Trachea

The purpose of this study was to examine the effect of the anticholinesteraseagent, soman, on macromolecular secretion by ferret trachea,in vitro We mounted pieces of ferret trachea in Ussing-typechambers. Secreted sulfated macro-molecules were radiolabeledby adding 500 µCi of ³³SO₄ to the submucosal medium andincubating for 17 hr. Soman added to the submucosal side produceda concentration-dependent increase in radiolabeled macromolecularrelease with a maximal secretory response (mean ± SD)of 202 ± 125% (n = 8) relative to the basal secretionrate at a concentration of 10^–7 m. The addition of either10^–6 M pralidoxime (acetyicholinesterase reactivator)or 10^–7 M atropine blocked the response to 10^–7m soman. At soman concentrations greater than 10^–7 M,secretion rate decreased and was not significantly differentfrom basal secretion. Additional experiments utilizing acetylcholineand the acetyicholinesterase inhibitor, physostigmine, suggestthat inhibition of secretion by high concentrations of somanmay be due to a secondary antagonistic effect of soman on muscannicreceptors.     

Dermal Absorption of Phthalate Diesters in Rats

Dermal Absorption of Phthalate Diesters in the Rat. ELSISI,A. E., CARTER, D. E., AND SIPES, I. G. (1989). Fundam Appl.Toxicol. 12, 70–77. This study examined the extent ofdermal absorption of a series of phthalate diesters in the rat.Those tested were dimethyl, diethyl, dibutyl, diisobutyl, dihexyl,di(2-ethylhexyl), diisodecyl, and benzyl butyl phthalate. Hairfrom a skin area (1.3cm in diameter) on the back of male F344rats was clipped, the [¹⁴C]phthalate diester was applied ina dose of 157 µmol/kg, and the area of application wascovered with a perforated cap. The rat was restrained and housedfor 7 days in a metabolic cage that allowed separate collectionof urine and feces. Urine and feces were collected every 24hr, and the amount of [¹⁴C] excreted was taken as an index ofthe percutaneous absorption. At 24 hr. diethyl phthalate showedthe greatest excretion (26%). As the length of the alkyl sidechain increased, the amount of [¹⁴C] excreted in the first 24hr decreased significantly. The cumulative percentage dose excretedin 7 days was greatest for diethyl, dibutyl, and diisobutylphthalate, about 50–60% of the applied ¹⁴C; and intermediate(20–40%) for dimethyl, benzyl butyl, and dihexyl phthalate.Urine was the major route of excretion of all phthalate diestersexcept for diisodecyl phthalate. This compound was poorly absorbedand showed almost no urinary excretion. After 7 days, the percentagedose for each phthalate that remained in the body was minimaland showed no specific tissue distribution. Most of the unexcreteddose remained in the area of application. These data show thatthe structure of the phthalate diester determines the degreeof dermal absorption. Absorption maximized with diethyl phthalateand then decreased significantly as the alkyl side chain lengthincreased.     

Cytotoxic Effects of Cyclophosphamide in the Mouse Seminiferous Epithelium: DNA Flow Cytometric and Morphometric Analysis

Cytotoxic Effects of Cyclophosphamide in the Mouse SeminiferousEpithelium: DNA Flow Cytometric and Morphometric Analysis. TOPPARI,J., BISHOP, P. C., PARKER, J. W., AHMAD, N., GIRGIS, W., ANDDIZEREGA, G. S. (1990). Fundam. Appl. Toxicol. 15, 44–52.Stage-specific cytotoxicity of cyclophosphamide (CP) in themouse testis was analyzed by quantitative DNA flow cytometryand morphometry. In five series of experiments, three mice wereinjected (ip) with either a single dose of CP at 30 mg/kg ora vehicle saline. At 3, 11, and 20 days later, spermatogeniccells from stages II–V, VII–VIII, and IX–XIof the seminiferous epithelial cycle were quantified by flowcytometry and by morphometry. CP killed a part of the differentiatingspermatogonia which became apparent at 11 days when the numberof pachytene spermatocytes at stages II–V and VII–VIIIwas decreased. At stages IX–XI, the number of leptoteneand zygotene spermatocytes declined at this time point. Thesedamages could be detected by morphometry, while flow cytometryshowed only the trend of the difference. At 20 days, both methodsrevealed a decrease in the number of haploid spermatids at stagesVII–VIII (48 and 33% decrease detected by morphometryand flow cytometry, respectively). DNA flow cytometry provedto be a rapid and practical method to detect stage-specificdisruption of spermatogenesis, while morphometry was more sensitive.