The Pulmonary Response and Clearance of Ludox Colloidal Silica after a 4-Week Inhalation Exposure in Rats

Rats were exposed to Ludox colloidal silica (CS) at concentrationsof 0, 10, 50, and 150 mg/m³ for 6 hr/day, 5 days/week for 4weeks. Rats were killed after 4 weeks of exposure and 10 daysor 3 months post exposure (PE). The exposure concentration of10 mg/m³ Ludox CS is considered to be the no-effect concentration.There were no exposure-related clinical signs in any group.After 4 weeks exposure, lung weights were increased significantlyin rats exposed to 50 and 150 mg/m³ Ludox CS, but lung weightswere similar to those of controls at 3 months PE. After 4 weeksexposure to 50 mg/m³ Ludox CS, a slight alveolar macrophageresponse, polymorphonuclear leukocytic infiltration, and TypeII pneumocyte hyperplasia in alveolar duct regions were present.After 3 months PE, these pulmonary lesions had almost disappearedwith removal of most dust-laden alveolar macrophages (AMs).The pulmonary response to 150 mg/m³ Ludox CS was similar incharacter but increased in magnitude from that seen at 50 mg/m³At 3 months PE, most particleladen AMs had disappeared and theremaining AMs were aggregated and sharply demarcated. A fewaggregates of particle-laden AMs appeared to transform intosilicotic nodules comprising macrophages, epithelioid cells,and lymphocytic infiltration in some animals. Some silicoticnodules showed reticular fiber networks with minute collagenfiber deposition. Tracheobronchial lymph nodes were enlargedwith aggregates of particle-laden AMs and hyperplastic histiocyticcells. Lung-deposited Ludox cleared rapidly from the lungs withhalf-times of approximately 40 and 50 days for the 50 and 150mg/m³ groups, respectively.     

Metallothionein Induction and Pulmonary Responses to Inhaled Cadmium Chloride in Rats and Mice

Inhaled CdCl₂ is a pulmonary carcinogen in rats but not in mice.We hypothesized that pulmonary metallothionein (MT) inductionmay be different in both species and thereby may lead to differentlevels of protection from Cd-induced lung injury. Fisher-344rats and B6C3F₁ mice were exposed for 4 weeks to CdCl₂ aerosolsof 0, 30, 50, and 150 µg/m³ air or 0, 10, 30, and 100µg/m³ air, respectively. Animals from each exposure groupwere terminated at 1, 30, and 133 days after the end of exposure.The lungs were lavaged for cell and biochemical analyses. Cadmiumand MT in lavagate and lung tissue were measured. The retentionhall-time of pulmonary Cd was greater in mice (290 vs 90 days,p<0.05). Cd exposure provoked an inflammatory response whichwas dose-dependent in both species, and while it was only short-livedin rats, it persisted throughout the observation period in miceat the high exposure concentrations. Mice were found to havea greater baseline level of MT (18.046.96 vs 11.71.98 µgMT/g control lung, p<0.05). Mice showed greater inducibilityof MT for a given CdCl₂ exposure concentration; however, bothspecies had a similar relationship between retained pulmonaryCd and MT induction though mice maintained increased MT levelsfor a longer period of time. The greater pulmonary baselineMT together with the longer presence of Cd-induced pulmonaryMT may result in greater protection from Cd carcinogenicityin spite of the greater pulmonary Cd-induced inflammation inmice.     

Acute, Subacute, and Subchronic Inhalation Toxicity Studies of Respirable Polymeric Methylene Diphenyl Diisocyanate (Polymeric MDI) Aerosol in Rats

Short-term inhalation toxicity studies with respirable polymericmethylene diphenyl diisocyanate (polymeric MDI) aerosol wereperformed in rats. The 4-hr LC50 was found to be 490 mg polymericMDI/m³ (95.5% <4.3 µm). Exposure of (4-week-old) ratsto 0, 2.2, 4.9, or 13.6 mg polymeric MDI/m³ (95% < 5 µm)for 2 weeks resulted in mortality, severe growth retardation,and elevated lung weights at 13.6 mg/m³ at 4.9 mg/m³ slightgrowth retardation and slightly elevated lung weights were observed.A 13-week study with 6-week-old rats exposed to 0.35, 1.4, or7.2 mg polymeric MDI/m³ (95% < 5 µm) revealed transientgrowth retardation and a slightly increased number of pulmonaryalveolar macrophages occasionally accompanied by increased numbersof mononuclear cells and fibroblasts in alveolar septa onlyat 7.2 mg/ m³ In a second 2-week study with 4 or 6-week-oldrats exposed to 14.1 mg polymeric MDI/m³ (95% < 5 µm),4-week-old rats died earlier and in greater numbers than 6-week-oldrats. In a second 13-week study with 6-week-old rats, usingexposure concentrations of 0, 4.1, 8.4, and 12.3 mg polymericMDI/ m³ (95% < 5 µm) and including a 4-week recoveryperiod, 12.3 mg/ m³ induced mortality, growth retardation, severerespiratory distress, increased lung weights, degeneration andhyperplasia of the nasal epithelium, accumulations of macrophagesin the lungs and mediastinal lymph nodes, and focal inflammatorychanges in the lungs. Rats exposed to 8.4 mg/ m³ showed respiratorydistress, lower body weights in males, increased lung weights,and similar, but much less severe, histopathological changesin the respiratory tract and mediastinal lymph nodes. Most ofthe histopathological changes seen at the higher concentrationswere also seen at 4.1 mg/m³ but to a very minor degree and ina few rats only. At the end of the 4-week posttreatment periodthe microscopical changes in nose, lungs, and mediastinal lymphnodes were still present but generally to a much less degreethan at the end of the exposure period. It was concluded thatthe dose-effect curve for repeated exposures of rats to respirablepolymeric MDI is very steep, and that the "no-observed-adverse-effectlevel" of polymeric MDI was 1.4 mg/m³ the actual no-adverse-effectlevel being lower than but most probably very close to 4.1 mg/m³.     

Teratology of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in a Complex Environmental Mixture from the Love Canal

The organic phase of a leachate (OPL) from the Love Canal chemicaldump site contains more than 100 organic compounds including2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). The teratogenicpotential of OPL was determined in two inbred and one hybridmouse strain which differ in their sensitivity to aromatic hydrocarbon(Ah) receptor-mediated toxicity. OPL was orally administeredin corn oil on Days 6–15 of gestation to C57BL/6J mice(Ah^b/ Ah^b) at doses of 0, 0.1, 0.3, 0.5, and 0.7 g kg^–1day^–1 and to DBA/ZJ (Ah^d/Ah^d) females, which were matedwith either DBA/2J or C57BL/6J males, at 0, 0.5, 1, and 2.0g kg^–1 day^–1. In C57BL/6J mice, which express ahigh-affinity Ah receptor that avidly binds TCDD, the ED50'sof OPL for cleft palate and hydronephrosis were 0.44 and 0.11g OPL kg^–1 day^–1, respectively. Maternal mortalitywas 5% at the highest dose. In DBA/2J fetuses, which expressa low-affinity receptor, neither treatment-related cleft palatenor hydronephrosis was induced by dose levels that caused 36%maternal mortality. In hybrid D2B6F1 fetuses, the incidenceof cleft palate reached only 8% at 2 g OPL kg^–1 day^–1but the ED50 for hydronephrosis was 0.76 g OPL kg^–1 day^–1.TCDD was similarly administered to pregnant C57BL/6J mice at0, 0.5, 1, 2, and 4 µg kg^–1 day^–1 and to DBA/2Jmice at 0, 0.5, 2, 4, and 8 µg kg^–1 day^–1.In C57BL/6J fetuses, the ED50's for cleft palate and hydronephrosiswere 4.6 and 0.73 µg TCDD kg^–1 day^–1, respectively.In DBA/2J fetuses the ED50's for cleft palate and hydronephrosiswere 15.0 and 6.4 µg TCDD kg^–1 day^–1, respectively.Both the OPL and TCDD caused maternal hepatomegaly and thymicatrophy in all strains, but increased only C57BL/6J fetal weights.OPL decreased the number of fetuses per C57BL/6J dam at thetwo highest doses but there were no other reproductive effectsin any of the groups. It was concluded that the OPL is teratogenicand that hydronephrosis is a sensitive measure of TCDD toxicityin a complex organic mixture. Based on the ED50's of OPL- andTCDD-induced cleft palate and hydronephrosis in the C57BL/6Jstrain, the OPL had TCDD equivalence of 6.6 and 10.5 ppm, respectively.These values compare closely with the chemical analysis of 3ppm. The results suggest that the teratologic effects are dueprimarily to the TCDD in the OPL and that these effects aremediated through the Ah receptor, but that the maternal thymicatrophy and hepatomegaly were due primarily to the non-TCDDcomponents of the OPL.     

Pulmonary Fibrosis Produced in F-344 Rats by Subchronic Inhalation of Aerosols of a 4000 Molecular Weight Ethylene Oxide/Propylene Oxide Polymer

Pulmonary Fibrosis Produced in F-344 Rats by Subchronic Inhalationof Aerosols of a 4000 Molecular Weight Ethylene Oxide/PropyleneOxide Polymer. KLONNE, D. R., DODD, D. E., Losco, P. E., TROUP,C. M., AND TYLER, T. R. (1988), Fundam Appl. Toxicol 10, 682–690.Inhalation of aerosols of the ethylene oxide/propylene oxidepolymer (U-5100) evaluated in this study has previously beenshown in acute and 2-week studies to produce toxicologic effectson the lungs, with increased lung weights and microscopic findingsof congestion and hemorrhage of pulmonary alveolar capillariesand necrosis of alveolar epithelial cells (D. R. KLONNE, D.J. NACHREINER, D. E. DODD, P. E. Losco, AND T. R. TYLER, 1987,Fundam. Appl. Toxicol. 9, 7737–784). In the present studies,F-344 rats were exposed 6 hr/day, 5 day/week for 2 weeks toaerosols at mean concentrations of 0,0.9, or 5.0 mg/m³ or for13 weeks to mean concentrations of 0, 0.3, 1.1, or 5.2 mg/m³.Following the 2-week study, minimal multifocal hemorrhage andeosinophilic proteinaceous debris in alveoli were observed inthe 0.9 mg/m³ group; similar lesions plus alveolar cell necrosiswere found in the 5 mg/m³ group. In the 13-week study, the 5.2mg/m³ group had a slight decrease in body weight gain, whileincreases in absolute and/or relative lung weights occurredfor both the 1.1 and 5.2 mg/m³ groups at the end of the exposureregimen and at the end of a 5-week recovery period. Histologiclesions of the lungs occurred in all U-5100-exposed groups andconsisted of hemorrhage, alveolar histiocytosis, interstitialpneumonia, and multifocal fibrosis. The incidence and severityof the pulmonary lesions were concentration related. At theend of the 5-week recovery period, there was little change inthe severity or incidence of the pulmonary lesions in the 1and 5 mg/m³ groups when compared to rats killed the day afterthe termination of exposures. In conclusion, exposure to aerosolsof U-5100 for 13 weeks produced generally slight but biologicallysignificant pulmonary fibrosis in rats at all the exposure concentrationstested in this study.     

Use of Lung Toxicity and Lung Particle Clearance to Estimate the Maximum Tolerated Dose (MTD) for a Fiber Glass Chronic Inhalation Study in the Rat

Short–term toxicity and lung clearance were assessed inrats exposed by inhalation to size-selected fibrous glass (FG)for 13 weeks. Results from this study and from a recent FG chronicinhalation study are presented here as guidelines for the selectionof a maximum tolerated dose (MTD) for chronic inhalation studiesof fibers. Fischer 344 rats were exposed using nose–onlyinhalation chambers, 6 hr/day, 5 days/week, for 13 weeks toone of five concentrations of FG (36, 206, 316, 552, or 714fibers/cc; expressed gravirnetrically, 3, 16, 30, 45, or 60mg/m³) or to filtered air. Rats were then held for an additional10 weeks of postexposure recovery. Test fiber was size–selectedfrom glass wool having a chemical composition representativeof building insulation. Rats were terminated at 7, 13, 19, and23 weeks after the onset of exposure to evaluate pulmonary pathology,lung epithelium cell proliferation, lung fiber burden, and lunglavage cells and chemistry. The effect of fiber inhalation onlung clearance of innocuous microspheres was also evaluated:following fiber exposure, six rats/group were exposed to ⁸⁵Sr–labeled3.0-µm polystyrene microspheres by intratracheal inhalationand then monitored for whole–body radioactivity duringthe 10–week recovery period. Data from the short–termstudy support the choice of 30 mg/m³ as the MTD for the previouschronic FG study and also provide indicators of long–termlung toxicity and functional impairment that can be used toestimate the MTD for future chronic fiber inhalation studies.     

The Disposition of Coal Dusts in the Lungs and Tracheobronchial Lymph Nodes of Dogs

The Disposition of Coal Dusts in the Lungs and TracheobronchialLymph Nodes of Dogs. Morrow, P.E. and Yuile, C.L. (1982). Fundam.Appl. Toxicol. 2:300–305. The pulmonary disposition, histopathologyand lymphatic uptake of anthracite (Tamaqua) and bituminous(Lower Kittaning) coal dusts were measured as part of a pulmonaryretention study which revealed a mean half-time of 1.92 yearsin dogs (Morrow et al. 1981). After brief (1-2.5 hr) exposuresto either natural or neutron-activated coals having an averageairborne mass concentration of {small tilde}90 mg m^–3and a 1.8 µm mass median aerodynamic diameter (g 2.5),dogs (n=12) were serially sacrificed up to 52 weeks after exposure.Coal dusts were found only in the lungs and pulmonary lymphnodes. The coals were considered indistinguishable as to theirpulmonary clearance and disposition and lymphatic uptake. Allcoals in the lung were associated mainly with the peribronchiolarand perivascular lymphatics or connective tissue spaces, andsome were found in alveolar macrophages. The lymphatic uptakeof coal dusts followed the powder function 0.55 t^0.613 wheret is in weeks and uptake is expressed as percent of the initialalveolar burden. In terms of pulmonary dust clearance, only4 percent of the initial alveolar burden appeared to have beentranslocated to the tracheobronchial lymph nodes in the first50 weeks, but this constituted {small tilde}14 percent of thetotal alveolar clearance. Histopathologically, one distinctionwas found: animals exposed to the highest level of neutron-activatedanthracite showed patchy hyaline thickening of some small bloodvessels and alveolar septa. The response was low grade, probablyexposure-related, but otherwise unremarkable.     

Clearance of Diesel Soot Particles from Rat Lung after a Subchronic Diesel Exhaust Exposure

Clearance of Diesel Soot Particles from Rat Lung after a ChronicDiesel Exhaust Exposure. Griffis, L.C., Wolff, R.K., Henderson,R.F., Griffith, W.C., Mokler, B.V. and McClellan, R.O. (1983).Fundam. Appl. Toxicol. 3:99-103. The participate exhaust ofdiesel engines consists of 0.1–0.2 µm mass mediandiameter particles composed of a carbonaceous core and adsorbedorganic compounds. A technique was needed to determine accumulatedlung burdens of particles in animals exposed to diesel exhaustas a determinant of dose. A method was developed for determininglung burdens of diesel soot particles in rats at 1 day, andat 1, 5, 15, 33 and 52 weeks after cessation of a subchronicexposure to diluted diesel exhaust. Lung tissue was dissolvedin tetramethylammonium hydroxide and the diesel soot separatedby centrifugation. The soot was suspended in water by sonicationand the light absorption of samples was compared to standardsuspensions of diesel soot. Recovery from lungs spiked with50–1000 µg of soot was 89±5%. Rats exposedover a period of 18 weeks to diluted diesel exhaust at averagenet diesel particle concentrations of 150, 940 and 4100 µg/m³had lung burdens of 35,220 and 1890 µg/g lung, respectively,one day after the last exposure. The long-term clearance ratesof soot had estimated half-times of 87±28, 99±4days, for the low and medium exposure groups, respectively.The clearance half-time for the high level exposure group of165 ± 8 days was significantly longer (P < 0.0001)than those of the other two groups.     

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.     

The Effects of in Vitro and Aerosol Exposures to Cadmium on Phagocytosis by Rat Pulmonary Macrophages

The Effects of in Vitro and Aerosol Exposures to Cadmium onPhagocytosis by Rat Pulmonary Macrophages. GREENSPAN, B. J.,and MORROW, P. E (1984). Fundam. Appl. Toxicol. 4, 48–57.Aerosol exposures of rats were performed to assess the in vivoeffects of cadmium on the phagocytosis of latex particles bypulmonary macrophages. An in vitro assay for particle uptakewas devised which allowed quantification of phagocytosis byadhering and nonadhering macrophages. In vitro exposure to CdCl₂caused dose-dependent decreases in viability (trypan blue exclusion),percentage cells with particles, total number of particles phagocytized,and the ability of the macrophages to adhere to a siliconizedglass surface. In vivo effects were studied following 30-minaerosol exposures to 1.5 mg/m³ Cd (MMAD = 0.35 µm, _g =1.45) or 5.0 mg/m³ Cd (MMAD = 0.45 µm, _g = 1.60) as CdCl₂.Phagocytic activity in the in vitro test system was increasedimmediately and at Day 1 in the low exposure group. However,following exposure to 5.0 mg/m³ Cd, phagocytic activity wasdepressed until 8 days postexposure. The results show that cadmiumis capable of modifying the phagocytic ability of macrophagesin vivo as well as in vitro. Determinations of the total numberof particles phagocytized were found to be more sensitive thanpercentage cells phagocytizing in detecting the effects of cadmiumexposure.     

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.     

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.     

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.     

Acute and 2-Week Inhalation Toxicity Studies on Aerosols of Selected Ethylene Oxide/Propylene Oxide Polymers in Rats

The ethylene oxide/propylene oxide (EO/PO) polymers evaluatedin this study have previously been shown to have a low orderof toxicity and/or irritancy by ocular, dermal, or oral routesof administration. These studies evaluated the acute inhalationtoxicity of respirable aerosols of three EO/PO compounds (U-660,U-2000, and U-5100) that differ in chain length, molecular weight,and viscosity. The respective 4-hr LC50 values (95% confidencelimits) for U-660, U-2000, and U-S 100 in Wistar albino ratswere 4670 (4090–5320), 330 (227–480), and 106 (45–245)mg/m³. Occasionally, slight increases in respiration rate andslight hyperactivity were observed during the postexposure period.All deaths were delayed for 2–5 days postexposure. Bodyweight gains were transiently depressed in rats exposed to U-2000and U-5100. Discolored lungs and livers occurred in animalswhich died during the 14-day postexposure period. Subsequently,a repeated-exposure study was conducted on U-5100 in F-344 ratsexposed for 6 hr/day, 5 days/week, for 9 exposures at mean concentrationsof 0, 5, 26, and 50 mg/m³ Portions of the control and 50 mg/m³groups were maintained for an additional 2-week recovery period.Exposure-related effects included transient urogenital wetnessin 50 mg/m³ group females; decreased body weight gain (7–29%)in all U-5100 groups except the 5 mg/m³ group females; increasesin absolute (17–52%) and relative lung weights in allU-5100 groups; macroscopic red foci in the lungs; and microscopicfindings of congestion and hemorrhage of pulmonary alveolarcapillaries and necrosis of alveolar epithelial cells. Lungweights remained elevated after the 2-week recovery period,but the severity of the microscopic lesions was noticeably less,indicating partial reversibility of the lesions. In conclusion,EO/PO polymers have a higher order of toxicity by inhalationin comparison to other routes of administration, vary considerablyin their acute lethal toxicity as a function of chain length/molecularweight, and induce pulmonary hemorrhage, and possibly edema,following repeated aerosol exposures at concentrations as lowas 5 mg/m³.     

Preclinical Toxicology Studies with Acyclovir: Genetic Toxicity Tests

Preclinical Toxicology Studies with Acyclovir: Genetic ToxicityTests. Clive, D., Turner, N.T., Hozier, J., Batson, A.G. andTucker, W.E., Jr. (1983). Fundam. Appl. Toxicol 3: 587–602.Acyclovir (ACV), an antiviral drug active in the treatment oforal and genital Herpes infections, has been evaluated for mutagenicand carcinogenic potential in a battery of in vitro and in vivoshort-termassays. Negative results were obtained in the following in vitrotests: Ames Salmonella, plate incorporation and preincubationmodification assays; E. coli polA⁺/polA^– DNA repair; yeast(S. cerevisiae D4) gene conversion; Chinese hamster ovary cells(HGPRT, APRT loci and ouabain-resistance marker); L5178 Y mouselymphoma cells (HGPRT locus and ouabain-resistance marker);and C3H/10Tmouse fibroblast neo-plastic transformation assay.All except the last assay were performed in the presence andabsence of an exogenous metabolic activation system. ACV waspositive at high concentrations x exposure times in the absenceof exogenous metabolic activation in the following in vitrosystems and at the indicated concentrations: BALB/c-3T3 neoplastictransformation (50 /µg/mL, 72 h exposure); human lymphocytecytogenetics (250–500 µg/mL, 48 h exposure); andL5178Y mouse lymphoma cells (TK locus, 400–2400 µg/mL,4 h exposure; predominantly small colony mutants of chromosomalorigin produced). No effects were seen in vivo (mouse dominantlethal assay; rat and Chinese hamster bone marrow cytogenetics)at up to maximum tolerated doses (MTD). An unusual clastogeniceffect was seen in Chinese hamsters at 5 times the MTD. Overall,positive effects were seen only at either high concentrations(250 µg/mL in vitro or plasma levels) or prolonged exposure(72 hr in the BALB/ c-3T3 neoplastic transformation assay).These studies support the view that ACV is a chromosomal mutagen,i.e., one which causes multi-locus damage but not single geneeffects. The significance of these results for the genetic riskof ACV to man is discussed.     

Kinetics of Respiratory Tract Absorption and Plasma Clearance of Horseradish Peroxidase in Guinea Pigs

Kinetics of Respiratory Tract Absorption and Plasma Clearanceof Horseradish Peroxidase in Guinea Pigs. CONNER, M. W., CHAUDHURI,I., ROGERS, A. E., and AMDUR, M. O. (1985). Fundam. Appl. Toxicol.5, 99–104. Horseradish peroxidase (HRP) absorption acrossthe wall of the upper airway, monitored by the amount detectedin the blood, is used to measure epithelial damage by toxins.Full details of kinetics of absorption and blood clearance havenot been reported previously. We measured the kinetics underthe experimental conditions used in testing toxins. HRP wasadministered to guinea pigs either by intraarterial injectionof a 7.5 µg bolus (plasma clearance) or by intratrachealinstillation of 1 mg (respiratory tract absorption). Plasmaconcentrations were monitored for 60 min. Plasma concentrationsof HRP rose linearly with time after intratracheal instillation,reaching 236 ± 51 ng/ml (mean ± SE) at 60 minafter instillation. HRP was cleared from the plasma rapidlyafter bolus injection. The elimination coefficient, k2, determinedfrom the biphasic log normal plot, was 0.322 min^–1. Thesedata were used to estimate the kinetics of absorption acrossthe respiratory epithelium. A single 3-hr exposure to an atmospherecontaining 2.5 mg/m³ of submicrometer zinc oxide particles increasedplasma concentration of HRP after intratracheal deposition (407± 63 ng/ml at 60 min) and had no effect on plasma clearance(k² = 0.342 min^–1). Therefore plasma concentrations ofHRP measured after intratracheal deposition can be used as asensitive indicator to evaluate the effects of inhalation ofa test atmosphere on epithelial permeability, if plasma clearancekinetics are not altered by the exposure.     

Kinetics of Hemoglobin and Albumin Adducts in Rabbits Subchronically Exposed to Benzo[a]pyrene

Benzo[a]pyrene (BaP) can form adducts with proteins after activationto a diolepoxide. Benzo[a]pyrene-hemoglobin and BaP-albuminadducts were measured in rabbits exposed to 0.5 or 5 µmol·kg^–1·week^–1for a total of 11 weeks (last injection on Day 75). Each dosegroup of nine rabbits was divided into three equal subgroups.For each dose, one subgroup received a single weekly injection(Mondays), the second had two equal weekly injections (Mondaysand Thursdays), and the third had five weekly injections (Mondaysthrough Fridays). Blood was collected prior to injection onDays 0, 7, 14, 21, 28, 35, 42, 49, 56, 77, 78, 80, 84, 92, 108,and 140 for adducts determinations, with Day 0 being a Monday.The measured concentration of hemoglobin adducts was independentof the frequency of administration for a given total weeklydose giving support to its value as a "biointegrator." In addition,animals injected with 0.5 and 5 µmol BaP·kg^–1·week^–1had respective mean adduct concentrations of 0.3 and 3 pmol/ghemoglobin. The blood concentration of albumin adducts was relatedto the frequency of injection with the animals receiving one,two and five injections/week having the lowest, intermediate,and highest adduct concentrations, respectively. Animals injectedwith 0.5 and 5 µmol BaP·kg^–1·week^–1had respective mean adduct concentrations of 5 and 20 pmol/gwhich are 17 and 7 times higher than their corresponding hemoglobinadducts' values. The corresponding albumin adducts' half-livescalculated from the day of cessation of exposure were 5.8 and9.6 days, compared with a reported 5.7 days for the half-lifeof the intact protein. Comparison of the pattern of hemoglobinadduct formation and removal with currently proposed kineticadduct model could only be performed for the 5 µmol BaP·kg^–1·week^–1dose as hemoglobin adduct concentrations fell below detectionlimit after cessation of exposure at the lower dose. The higherdose data suggested that the model reasonably describe the overallprofile observed. However, this limited comparison also suggestedthat, for hydrophobic substances such as BaP capable of enzymaticinduction, introduction of both an induction function and considerationof the kinetics of the parent compound itself in the organismwould probably improve the fit.     

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.     

Pulmonary Retention of Inhaled Diesel Particles after Prolonged Exposures to Diesel Exhaust

Pulmonary Retention of Inhaled Diesel Particles after ProlongedExposures to Diesel Exhaust CHAN, T. L., LEE, P. S., AND HERING,W. E. (1984). Fundam. Appl. Toxicol. 4, 624–631. The effectof continuous exposure to diluted diesel exhaust on the pulmonaryretention of inhaled diesel particles was studied in male Fischer344 rats. Test animals were first exposed to clean air or diluteddiesel exhaust in exposure chambers at nominal particulate concentrationsof 250 µg/m³ or 6 mg/m³ for 20 hr/day, 7 days/week, forperiods lasting from 7 to 112 days, followed by a nose-onlyexposure to ^l4C-tagged diesel particles for 45 min. At preselectedtime intervals after the radioactive exposure, the ¹⁴C-activitiesin the lungs of groups of four animals were measured to determinethe clearance of the ^l4C-diesel particles up to 1 year. Thepulmonary retention of the radioactive diesel particles wasgreater in animals which had been preexposed to diesel exhaustThe slower alveolar clearance of particle-laden macrophagesand leukocytes can be described by a normal Diphasic clearancemodel. Since some of the macrophages were found sequesteredas aggregates in the pulmonary region, a slow-clearing residualcomponent was included in a modified lung retention model. Whenthese residual fractions were determined and excluded from theactive particulate transport within the lungs, normal alveolarclearance rates were calculated for the animals with a preexposurediesel particulate dose less than 0.8 mg. Slower clearance wasobserved at a dose of 6.5 mg and no clearance was evident ata dose of 11.8 mg in their lungs. In effect, the greater retentionof inhaled particles can be interpreted as-a sign of impairedlung clearance attributable to the prolonged exposures to highconcentrations of diesel exhaust gases and/or the presence ofaccumulated carbonaceous particles residing in sequestered macrophageaggregates in the lungs.     

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.