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.     

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.     

Potentiation of Bromotrichloromethane Hepatotoxicity and Lethality by Chlordecone Preexposure in the Rat

Potentiation of Bromotrichloromethane Hepatotoxicity and Lethalityby Chlordecone (Kepone®) Preexposure in the Rat. Agarwal,A.K. and Mehendale, H.M. (1982). Fundam. Appl. Toxicol. 2:161-167.These studies were designed to provide dose-response relationshipsfor chlordecone (CD) potentiation of BrCCl₃ hepatotoxicity inmale rats using biochemical, functional and histopathologicalparameters. The influence of this interaction on BrCCl₃ lethalitywas also examined. Male Sprague-Dawley rats (175–200g)received a single ip dose of 1, 5,10,15 or 25 µL BrCCl₃/kgfollowing a 15 day dietary pretreatment of 0 or 10 ppm CD. Twentyfour hrs after BrCCl₃ challenge, biliary excretion of phenolphthaleinglucuronide (PG), bile flow, serum transaminases (SGOT and SGPT),serum ICD and OCT were examined as functional and biochemicalindices of hepatic injury. Effect of CD on 48 hr LD₅₀ of BrCCl₃was also examined using the method of moving averages. Withthe exception of 1 µL BrCCl₃/kg dose which had no effect,CD-BrCCI₃ combination resulted in potentiation of hepatotoxicityby all parameters examined. Activity of all the serum enzymeswas elevated in a dose related manner. A dose related decreasein the biliary excretion of PG and bile flow was observed. Theseeffects were more pronounced at the higher doses of BrCCl₃.Extensive centrilobular necrosis was observed in the animalsgiven CD-BrCC₃ combination and the necrogenic effect was moresevere at the doses of 15 µL and 25 µL BrCCl₃/kg.BrCC₃-lethality was increased 5-fold by CD as indicated by thedecreased LD₅₀- The results suggest that CD-induced BrCCl₃ toxicityis manifested both in the form of hepatotoxicity and lethalityand since the hepatic functional status is greatly compromised,the CD potentiatedhepatic failure is related to lethality.     

Fractional Hepatic Localization of 14CHCl3 in Mice and Rats Treated with Chlordecone or Mirex

Fractional Hepatic Localization of ¹⁴CHCl₃ in Mice and RatsTreated with Clordecone or Mirex. Hewitt, L.A., Hewitt, W.R.,and Plaa, G.L. (1983). Fundam. Appl. Toxicol. 3:489–495.In rodents chlordecone, but not mirex, a nonketonic structuralanalog, significantly augments CHCl₃-induced liver damage, atleast in part, by increasing CHCl₃ bioactivation. To determinewhether the fractional distribution of CHCl₃ was altered inchlordecone-pretreated animals, the irreversible binding of¹⁴CHCl₃ to various liver constituents (a measure of CHCl₃ bioactivation)was examined in vivo in mice and rats. Chlordecone, but notmirex, increased both total and irreversibly bound ¹⁴CHCl₃;furthermore, changes in the ¹⁴Clocalization between lipid, proteinand acid-soluble fractions were noted. Thus, the results suggestthat differences exist between chlordecone and mirex with respectto their capacity to increase the quantity of CHCl₃-derivedreactive metabolite and the eventual distribution of reactivemetabolite.     

Metabolism of Acrylic Acid to Carbon Dioxide in Mouse Tissues

Acrylic acid (AA) is acutely irritating at sites of initialcontact but causes little systemic toxicity probably due toits rapid metabolism to CO₂ and acetyl-CoA via a secondary pathwayof propionic acid catabolism. In this study, the rate of AAoxidation in 13 tissues of C3H mice was measured by incubatingtissue slices with [1-¹⁴C]AA and collecting ¹⁴CO₂. Oxidationof AA followed pseudo-Michaelis-Menten kinetics in the liver,kidney, and skin. Pseudo-K_m values were similar among thesetissues and averaged 0.67 mM. The maximal rate of AA oxidationin kidney, liver, and skin was 2890±436 (mean±SE,N=3), 616±62, and 47.9±5.8 nmol/hr/g, respectively.The remaining organs oxidized AA at rates less than 40% of therate in liver. Rates of metabolism in tissues from male andfemale mice were similar. 3-Hydroxypropionic acid was the onlymetabolite detected by high-performance liquid chromatographicanalysis following incubation of tissues with [1-¹⁴C]AA. Kidneyand liver also oxidized [2,3-¹⁴C]AA and [1-¹⁴C]acetate well,thus providing for the complete metabolism of AA carbons toCO₂. These results demonstrate that the rate of AA metabolismvaries significantly among mouse tissues and suggest that thekidneys and liver are major sites of detoxification of AA.     

Potentiation of CCl4 and CHCl3 Hepatotoxicity and Lethality by Various Alcohols

Potentiation of CCl₄ and CHC1₃ Hepatotoxicity and Lethalityby Various Alcohols. RAY, S. D., AND MEHENDALE, H. M. (1990).Fundam. Appl. Toxicol. 15, 429–440. Various aliphaticalcohols potentiate the toxicity of a wide range of xenobioticsincluding several haloal-kanes. The present series of experimentswere designed to test: (i) whether a single subtoxic dose ofalcohol can potentiate CCl₄, and CHCl₃ hepatotoxicity, and (ii)whether this potentiation leads to greater animal lethality.Selected members of a homologous series of straight chain alcoholswere chosen for this study. Methanol, ethanol, isopropanol,t-butanol, pentanol, hexanol, octa-nol, decanol, and eicosanolat equimolar doses (10 mmol/kg) were tested in the present investigation.Each alcohol was administered orally to male Sprague-Dawleyrats (175–250 g) 18 hr prior to a single oral administrationof CCl₄ or CHCl₃. Liver injury was assessed by plasma transaminases(alanine aminotransferase, ALT; aspartate aminotransferase,AST) and histopathological examination of liver sections 24hr after the halomethane treatment. None of these alcohols aloneincreased plasma ALT or AST significantly, whereas CCl₄or CHCl₃administration to alcohol-treated animals resulted in significantelevation of plasma transaminases. Eicosanol (20-carbon alcohol)did not potentiate the toxicity of either halomethane. Methanol,ethanol, isopropanol, and decanol in combination with CCl₄ causedmassive liver damage but failed to augment CCl₄ lethality. t-Butanol,pentanol, hexanol, and octanol significantly decreased the LD50of CCl₄. The hepatotoxic effects of CHCl₃ were potentiated byall of the alcohols and the LD50s were also decreased significantly.On a comparative basis, alcohol-potentiated CHCl₃ toxicity wasgreater than the toxicity of CCl₄. These findings indicate thateven though halomethane liver injury might be potentiated byalcohols, the underlying mechanisms differ among alcohols sincenot all alcohols potentiate the lethal effects of these halomethanes.     

4,4'-Methylenebis(2-chloroaniline) (MOCA): The Effect of Multiple Oral Administration, Route, and Phenobarbital Induction on Macromolecular Adduct Formation in the Rat

The effect of multiple oral administration of MOCA, a suspecthuman carcinogen, was studied in the adult male rat. As manyas 28 consecutive daily doses of [¹⁴C]MOCA at 28.1µmol/kgbody wt (5 µC1/day) were administered and rats were euthanizedat weekly intervals for 7 weeks. MOCA adduct formation for globinand serum albumin was evaluated by determination of [¹⁴C]MOCAcovalent binding. The covalent binding associated with globinshowed a linear increase over the 28-day exposure period with342 fmol/mg globin 24 hr after the final dose. More extensivecovalent binding was detected for albumin with 443 fmol/mg albuminafter the final dose, but increases were not linear. After cessationof dosing, the albumin adduct levels decreased rapidly (t _1/2=4.6 days) in relation to globin adduct levels (t _1/2 =16.1days). The MOCA-globin adduct t _1/2 is consistent with thatdetermined after a single 281 µmol/kg oral dose of MOCA.Significant differences related to route of administration weredetected for 24-hr globin covalent binding with ip > po >dermal. Distribution of undifferentiated [¹⁴C]MOCA was highestin the liver at 24 hr with tissue levels for liver > kidney> lung > spleen > testes > urinary bladder. Inductionof cytochrome P450 enzymes by administration of phenobarbital(100 mg/kg/day/3 days) resulted in a significant (p < 0.05)increase in MOCA-globin adduct formation detected with 33.5pmol/ mg globin for induced rats versus 13.6 pmol/mg globinfor control rats. Although MOCA-globin and albumin adducts showdiffering stability, quantification of such MOCA adducts maybe useful for long-term industrial biomonitoring of MOCA.     

Chloroform Hepatotoxicity in the Mongolian Gerbil

Chloroform Hepatotoxicity in the Mongolian Gerbil. EBEL, R.E., BARLOW, R., and MCGRATH, E. A. (1987). Fundam. Appl. Toxicol.8, 207–216. CHCl₃ hepatotoxicity was studied in the maleMongolian gerbil and compared to that in the male Sprague–Dawleystrain rat. Based on elevations in serum transaminase activitiesin response to CHCl₃ exposure, control gerbils were more sensitiveto CHCl₃ than were gerbils treated with phenobarbital, chlordecone,mirex, or 3-methylcholanthrene. The increased sensitivity ofthe control relative to the induced gerbil was consistent withearlier observations of CCl₄ hepatotoxicity (Ebel, R. E., andMcGrath, E. A., 1984, Toxicol. Lett., 22, 205–210). Microsomalenzyme concentrations or activities were not decreased in thecontrol or induced gerbils in response to CHCl₃ exposures ofup to 200 µl/kg. At a dose of 500 µl/kg, cytochromeP-450 and its reductase were decreased by about 25% in the chlordecone-inducedgerbil. In contrast, chlordecone- and phenobarbital-inducedrats were sensitive to CHCl₃ as evidenced by marked elevationsin serum transaminase activities, decreases in microsomal enzymeconcentrations or activities, and a transient decrease in hepaticnonprotein sulfhydryl groups. Control rats were insensitiveto CHCl₃. Histopathological changes in the livers of these animalswere consistent with alterations in the biochemical parametersmeasured. The relationship between sensitivity to the hepatotoxiceffects of CHCl₃ and CCl₄ was different for the gerbil and rat.     

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.     

Sex Differences in Monochloroacetate Pretreatment Effects on Chloroform Toxicity in Rats

Previous studies have shown that dichloroacetate and trichloroacetateincrease the toxicity of CHCl₃. The present experiments weredesigned to determine if monochloroacetate (MCA) similarly affectsCHCl₃ toxicity. There were occasional differences, but overallkidney function indices (urine volume, osmolality and electrolyteconcentration, glucosuria, retention of urea nitrogen in plasma)were not affected differently at either 24 or 48 hr after CHCl₃in saline and MCA pretreated Sprague-Dawley rats of either sex.Males pretreated with MCA had 45-fold greater plasma alanineaminotransferase (ALT) compared to the saline pretreated groupsimilarly dosed with CHCl₃. ALT was increased threefold in femalerats, a modest change that suggests hepatic damage, and BUNwas nonsignificantly increased. Therefore hepatic and renalfunctions were assessed in females. MCA pretreatment did notalter the effects of CHCl₃ on hepatic excretory function orglomerular or tubular function. Bile production and glomerularfiltration were both decreased in the MCA group treated withpeanut oil, suggesting that MCA impairs both liver and kidneyfunction in female rats. MCA pretreatment increases CHCl₃ hepatoxicitymarkedly in male rats and only slightly in female rats. Thisdifference is likely due to the different effects, in malesand females, of MCA on the cytochrome P450 isoforms that activateCHCl₃. The effects of MCA on renal function in females woulddecrease CHCl₃ delivery to kidney cells, suggesting that MCAmay alter the distribution of CHCl₃.     

Nephrotoxic Interactions between Ketonic Solvents and Halogenated Aliphatic Chemicals

Nephrotoxic Interactions between Ketonic Solvents and HalogenatedAliphatic Chemicals. Hewitt, W. R., and Brown, E. M. (1984).Fundam. Appl. Toxicol. 4, 902–908. Recent studies haveindicated that (1) ketonic solvents and ketogenic chemicalscan potentiate the nephrotoxic and hepatotoxic effects of oneor more halogenated hydrocarbons; and (2) the relative abilityof ketones to potentiate the liver injury produced by chloroform(CHCl₃) may be influenced by the carbon skeleton length of theketone. Although five ketones (acetone, 2-butanone, 2-pentanone,2-hexanone, (HX), and 2-heptanone) increased CHCl₃-induced kidneyand liver injury in male, Fischer 344 rats, no relationshipbetween ketone chain length and potentiating capacity was observed.HX potentiated the CHCl₃-induced depletion of hepatic glutathionecontent and increased the irreversible binding of ^l4CHCl₃-derivedradiolabel to hepatic constituents. In contrast, CHCl₃ did notalter glutathione content in the renal cortex of either vehicle-or HX-pretreated rats. Although HX increased the binding of¹⁴C from ¹⁴CHCl₃ to renal cortical macromolecules, the magnitudeof the increase was unremarkable, approaching only the extentof hepatic ¹⁴C binding in vehicle-pretreated rats challengedwith ^l4CHCl₃. Since the severity of renal and hepatic injurywas comparable in rats receiving the combination of HX + CHCl₃,it appeared that HX potentiated CHCl₃ nephro- and hepatotoxicityby different mechanisms. Ketone pretreatment did not potentiatethe renal injury produced by potassium dichromate or hexachloro-l,3-butadiene.     

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.     

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     

Pretreatment with Drinking Water Solutions Containing Trichloroethylene or Chloroform Enhances the Hepatotoxicity of Carbon Tetrachloride in Fischer 344 Rats

Pretreatment with Drinking Water Solutions Containing Trichloroethyleneor Chloroform Enhances the Hepatotoxicity of Carbon Tetrachloridein Fischer 344 Rats. Steup, D. R., Wiersma, D., McMillan, D.A., and Sipes, I. G. (1991). Fundam. Appl. Toxicol. 16, 798–809.Previous studies have demonstrated that various compounds, includingthe common groundwater contaminants trichloroethylene (TCE)and chloroform (CHCl³), can produce a synergistic toxic responsewhen coadministered with the model hepatotoxicant carbon tetrachloride(CCI₄. This phenomenon has not, however, been demonstrated followingadministration of these compounds in drinking water. Initialexperiments indicated that Fischer 344 (F-344) rats were significantlymore sensitive to these effects than the more commonly utilizedSprague–Dawley strain. To establish the suitability ofthis strain as a model, a variety of indicators of hepatotoxicitywas evaluated and compared to histological evidence of injury24 hr after dosing with CCl₄ or a combination of CCl₄ + TCE.Plasma alanine aminotransferase (ALT) activity was the mostreliable indicator of hepatic injury and was well-correlatedwith the histologic data. Dose–response studies utilizingsimultaneous ip dosing confirm the sensitivity of the F-344rat, demonstrating synergistic toxicity at doses as low as 0.165mmol/kg of CCl₄ and 0.6 mmol/kg of TCE. Synergism was also detectedfollowing simultaneous ip administration of 1 mmol/kg CCl₄ and0.5 mmol/kg of CHCl₃. To evaluate the effects of drinking waterexposure, rats were pretreated for 3 days with solutions containingTCE (0–40 mM) or CHCl₃ (0–8 mM) stabilized with1% Emulphor (EL-620P) as their only source of fluids. A single,ip dose of CCl₄ (1 mmol/kg) was then administered and 24 hrlater animals were killed for examination of liver histologyand determination of ALT activity. Although none of the pretreatmentswere detectably hepatotoxic, rats which drank 15 and 40 mm TCEor 8 mm CHCl₃ exhibited an enhanced response to CCl₄     

Dieldrin Pretreatment Alters [14C]Dieldrin and [3H]7,12-Dimethylbenz[a]anthracene Uptake in Rainbow Trout Liver Slices

We previously demonstrated that pretreatment of rainbow troutwith the organochlorine insecticide dieldrin altered in vivodisposition of a subsequent [¹⁴C]dieldrin dose. This was notexplained by changes in total lipid content or the activityof common xenobiotic metabolizing enzymes. We hypothesized thatdieldrin induced hepatic proteins responsible for organochlorine(OC) sequestration, transport, or excretion and that these changesreflected an adaptive response of trout to OC exposure. Here,uptake of 1.18 µM [¹⁴C]-dieldrin by precision cut liverslices was increased by dieldrin pretreatment of rainbow trout.Uptake of 0.118 and 1.18 µM [³H]-7,12-dimethylbenz[a]anthracene(DMBA) and efflux of 0.118 µM [³H]DMBA were significantlyincreased in slices from dieldrin pretreated trout. Liver sliceuptake of 10 but not 1.18 µM [³H] estradiol and [³H]cholicacid was significantly increased by dieldrin pretreatment. Therewere no such significant differences for [³H]cholesterol, or[³H]cholesterol-oleate, or [³H]oleic acid uptake. Dieldrin pretreatmentdid not alter hepatic microsomal metabolism of [³H]DMBA or [¹⁴C]benzo[a]pyreneor content of six cytochrome P450 isozymes, as quantitated byWestern blot analysis. These results provide further evidencethat altered disposition of [¹⁴C]dieldrin and [³H]DMBA in dieldrin-pretreatedtrout was not explained by microsomal enzyme induction but reflectedaltered processes integral to hepatocellular transmembrane kinetics.These changes may have important implications for OC bioaccumulationby rainbow trout and demonstrate an interaction between dieldrinand DMBA in the absence of cytochrome P450 system induction.     

Stimulation of Prostaglandin Production by Quinolone Phototoxicity in Balb/c 3T3 Mouse Fibroblast Cells in Vitro

Sparfloxacin (SPFX) and levofloxacin (LVFX) with ultraviolet-A(UVA) irradiation have been reported to induce skin inflammationdue to phototoxicity in Balb/c mice. We examined the productionof arachidonic acid metabolites induced by quinolone phototoxicityin Balb/c 3T3 mouse fibroblast cells in vitro. The cells weresimultaneously treated with SPFX or LVFX at 1,10, or 100 µMand UVA irradiation for 5 min (0.5 J/cm²). They were then culturedin quinolone-free medium for 24 hr, and the concentrations ofprostaglandin E₂ (PGE₂ 6-ketoprostaglandin F₁ (6-keto-PGF₁),and leukotriene B₄ (LTB₄) in the incubation medium were measured.Furthermore, the effect of quinolone photoproducts on the productionof the inflammatory mediators and that of indomethacin on PGE₂level were also examined. Treatment with SPFX at 100 µMplus UVA irradiation markedly increased levels of PGE₂ and 6-keto-PGF₁but not that of LTB SPFX or LVFX alone at up to 100 µM,100 µM SPFX, or 100µM LVFX, or less plus UVA irradiation,or UVA-preirradiated quinolone up to 100µM had no effect.indomethacin even at 0.1 µM completely inhibited the PGE₂elevation induced by 100 µM SPFX with UVA. These resultssuggest that PGs released from dermal fibroblasts in the simultaneouspresence of quinolone and UVA could contribute in part to thedevelopment of skin inflammation in vivo.     

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.     

Hepatic Failure Leads of Lethality of Chlordecone-Amplified Hepatotoxicity of Carbon Tetrachloride

Hepatic Failure Leads to Lethality of Chlordecone-AmplifiedHepatotoxicity of Carbon Tetrachloride. SONI, M. G., AND MEHENDALE,H. M. (1993). Fundam. Appl. Toxicol. 21, 442–450. Chlordecone (Kepone) amplification of CCl₄ toxicity occurs atsmall, nontoxic levels of chlordecone and CC1₄ and results inhighly increased irreversible hepatotoxicity culminating inlethality. Although it is generally assumed that CCl₄ lethalityis due to hepatic failure, no definitive studies are availablein the literature bridging massive liver failure and death.The present studies were designed to evaluate whether hepaticfailure is the cause of the lethality during chlordecone-amplifiedCCl₄ toxicity. Male Sprague-Dawley rats were maintained on controlor a chlordecone (10 ppm) diet for 15 days and injected withCCl₄ (100 µl/kg, ip) on Day 16. Rats were killed at 0,6, 12, 24, 36, and 48 hr after CCl₄ challenge. Hepatic failurewas evaluated by measuring plasma glucose, ammonia, bilirubin,aspartate trans-aminase (AST), alanine transaminase (ALT), sorbitoldehydrogenase (SDH), hepatic ATP, glycogen, and by histologicaland histomorphometric analyses. Plasma creatinine, urea, andkidney histopathology were also assessed for possible renalinjury. As expected CCl₄ administration to chlordecone-pretreatedrats resulted in 20% lethality by 36 hr, which progressed withtime, and all rats died within 72 hr. A significant and progressivehypoglycemia was observed with a 60% reduction in plasma glucoseat 48 hr. Hepatic glycogen content dropped precipitously. Similarly,hepatic ATP levels remained suppressed (80% of control) at allthe time points studied. Plasma ammonia levels were significantlyelevated, and by 48 hr, a threefold increase was observed. PlasmaALT, AST, SDH, and bilirubin increased progressively until thedeath of rats receiving the chlordecone + CCl₄ combination.Histopathologically, the liver sections revealed a progressiveand irreversible damage evidenced by vacuolation, accumulationof fat, and increase in hepatocellular necrosis resulting indisrupted hepatolobular structure. Administration of the samedose of CCl₄ to rats maintained on a normal diet resulted inonly marginal changes in plasma enzymes, no increase in serumbilirubin, and no significant injury in liver sections at 24hr. CCl₄ administration to chlordecone-pretreated rats resultedin a marginal increase in plasma creatinine and urea only atlater time points. Histopathological examination of sectionsof kidneys from rats receiving either chlordecone + CCl₄ combinationor the same dose of CCl₄ alone did not reveal any evidence ofsignificant renal injury. The clinical end points of hepaticfunction measured in the present study are consistent with thesequel of hepatic failure and hepatic encephalopathy leadingto animal death in chlordecone-amplified CCl₄ toxicity.     

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.     

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.