Jon E. Dahl

RAT LUNG INFLAMMATORY RESPONSES AFTER IN VIVO AND IN VITRO EXPOSURE TO VARIOUS STONE PARTICLES

Rat lung alveolar macrophages and type 2 cells were exposed for 20 h in vitro to various stone particles with differing contents of metals and minerals (a type of mylonite, gabbro, feldspar, and quartz). The capability to induce the release of the inflammatory cytokines interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-alpha), and macrophage inflammatory protein-2 (MIP-2) was investigated. We found marked differences in potency between the various particles, with mylonite being most potent overall, followed by gabbro, and with feldspar and quartz having an approximately similar order of lower potency. The results also demonstrated differences in cytokine release pattern between the two cell types. For all particle types including quartz, type 2 cells showed the most marked increase in MIP-2 and IL-6 secretion, whereas the largest increase in TNF-alpha release was observed in macrophages. To investigate possible correlations between in vitro and in vivo inflammatory responses, rats were instilled with the same types of particles and bronchoalveolar lavage (BAL) fluid was collected after 20 h. The results demonstrated a correlation between the in vitro cytokine responses and the number of neutrophilic cells in the BAL fluid. The BAL fluid also showed a strong MIP-2 response to mylonite. However, this was the only particle type to give a significant cytokine response in the BAL fluid. We further examined whether a similar graded inflammatory response would be continued in type 2 cells and alveolar macrophages isolated from the exposed animals. Again a differential cytokine release pattern was observed between type 2 cells and macrophages, although the order of potency between particle types was altered. In conclusion, various stone particles caused differential inflammatory responses after both in vitro and in vivo exposure, with mylonite being the most potent stone particle. The results suggest the alveolar type 2 cell to be an important participant in the inflammatory response following exposure to particles.Rat lung alveolar macrophages and type 2 cells were exposed for 20 h in vitro to various stone particles with differing contents of metals and minerals ( a type of mylonite, gabbro, feldspar, and quartz). The capability to induce the release of the inflammatory cytokines interleukin-6 ( IL-6) , tumour necrosis factor-alpha (TNF-a ) , and macrophage inflammatory protein-2 (MIP-2) was investigated. We found marked differences in potency between the various particles, with mylonite being most potent overall, followed by gabbro, and with feldspar and quartz having an approximately similar order of lower potency. The results also demonstrated differences in cytokine release pattern between the two cell types. For all particle types including quartz, type 2 cells showed the most marked increase in MIP-2 and IL-6 secretion, whereas the largest increase in TNF-a release was observed in macrophages. To investigate possible correlations between in vitro and in vivo inflammatory responses, rats were instilled with the same types of particles and bronchoalveolar lavage (BAL) fluid was collected after 20 h. The results demonstrated a correlation between the in vitro cytokine responses and the number of neutrophilic cells in the BAL fluid. The BAL fluid also showed a strong MIP-2 response to mylonite. However, this was the only particle type to give a significant cytokine response in the BAL fluid. We further examined whether a similar graded inflammatory response would be continued in type 2 cells and alveolar macrophages isolated from the exposed animals. Again a differential cytokine release pattern was observed between type 2 cells and macrophages, although the order of potency between particle types was altered. In conclusion, various stone particles caused differential inflammatory responses after both in vitro and in vivo exposure, with mylonite being the most potent stone particle. The results suggest the alveolar type 2 cell to be an important participant in the inflammatory response following exposure to particles.

Testicular necrosis and DNA damage caused by deuterated and methylated analogs of 1,2-dibromo-3-chloropropane in the rat

To study the role of metabolism in 1,2-dibromo-3-chloropropane (DBCP)-induced testicular damage in rats, selectively deuterated and methylated analogs of DBCP were given as a single ip dose of 340 mumol/kg and testicular toxicity was determined 10 days after treatment. None of the four deuterated analogs C1-D2-, C2-D1-, C3-D2-, or C1-C2-C3-D5-DBCP reduced the degree of testicular damage compared to DBCP, indicating that metabolic cleavage of a C-H bond was not rate-limiting in DBCP-induced testicular toxicity. Of the five methylated analogs, C1-methyl-, C1-dimethyl-, C2-methyl-, and C3-methyl-DBCP and 1,2-dibromo-4-chlorobutane, only C3-methyl-DBCP caused testicular toxicity. DBCP treatment resulted in increased testicular DNA damage at doses of 85-170 mumol/kg as measured by alkaline elution of DNA from testicular cells isolated 3 hr after in vivo treatment. The perdeutero-DBCP analog induced testicular DNA damage that was at least as extensive as that induced by DBCP. Of the methylated analogs tested, only C3-methyl-DBCP gave a marked dose-dependent increase in testicular DNA damage between 170 and 540 mumol/kg. There were no significant differences in the testicular tissue distribution between DBCP, perdeutero-DBCP, and the methylated DBCP analogs. Furthermore, in distribution studies with DBCP, C1-methyl- and C3-methyl-DBCP, and 1,2-dibromo-4-chlorobutane, the highest tissue concentrations were found in the kidneys, followed by the liver and then the testes. The fact that testicular DNA damage of DBCP and its deuterated and methylated analogs paralleled their ability to cause testicular necrosis and atrophy makes measurement of DNA damage a very useful correlate in mechanistic studies of DBCP-induced testicular cell death.

Species differences in testicular necrosis and DNA damage, distribution and metabolism of 1,2-dibromo-3-chloropropane (DBCP)

The human testicular toxicant 1,2-dibromo-3-chloropropane (DBCP) was studied for the same end-point in 4 different species of laboratory animals. Marked necrosis and atrophy of the seminiferous epithelium were observed in rats and guinea pigs 10 days after a single i.p. administration of DBCP (170-340 mumol/kg), whereas significantly less damage was observed in hamsters and mice. The testicular concentrations of DBCP measured at various time-points after the i.p. injection of DBCP indicated that factors in addition to tissue concentration were of importance for the observed species differences in sensitivity towards DBCP-induced testicular damage. Also, there did not seem to be any direct correlation between DBCP-induced in vivo testicular toxicity and in vitro GSH-dependent dehalogenation, inasmuch as the rate of bromide release from DBCP with hamster testicular cytosol was as fast as that with rat cytosol. Testicular DNA damage, as determined by alkaline elution 60 min after in vivo administration of 170 mumol/kg DBCP, was observed only in rats and guinea pigs. Thus, induction of DNA damage correlates with the relative susceptibilities of the species towards DBCP-induced testicular necrosis. To further study species differences in testicular activation of DBCP to DNA-damaging intermediate(s), cells isolated from the testes of the 4 species were incubated with DBCP. Testicular cells from rats and guinea pigs were the only preparations developing substantial DNA damage after 60 min incubation with low concentrations of DBCP (5-50 microM). The findings indicate that rats are sensitive towards DBCP-induced testicular necrosis because rat testicular cells easily activate DBCP to a DNA-damaging intermediate(s). The relative high testicular DBCP concentration as well as the ability to activate DBCP may explain the sensitivity of guinea pigs towards DBCP-induced testicular toxicity.

Renal necrosis and DNA damage caused by selectively deuterated and methylated analogs of 1,2-dibromo-3-chloropropane in the rat

Selectively deuterated and methylated analogs of the nematocide 1,2-dibromo-3-chloropropane (DBCP) were compared to DBCP in causing acute renal damage in rats. All of the six deuterated analogs tested at 340 mumol/kg, including the perdeutero compound, failed to significantly alter the kidney necrosis observed at 48 hr compared to DBCP. Furthermore, when the perdeutero analog was administered at several doses (42.5, 85, 170, and 340 mumol/kg), it caused kidney damage that was not significantly different than that caused by an equivalent molar dose of nondeuterated DBCP. Of the five methylated analogs tested at 170 and 340 mumol/kg, only C3-methyl-DBCP and 1,2-dibromo-4-chlorobutane caused nephrotoxicity. The C2-methyl-, C1-dimethyl-, and C2-methyl-DBCP analogs failed to cause renal necrosis determined 48 hr after dosing. In distribution studies DBCP, perdeutero-DBCP, and all the methylated analogs were found to concentrate in the kidney approximately 25 times relative to plasma 1 hr after administration. DBCP at doses of 4.3 mumol/kg and higher caused DNA damage in the kidney as early as 10 min after administration, as measured by alkaline elution of DNA from isolated kidney nuclear preparations. Perdeuteration did not decrease the DNA damaging effect of DBCP. The ability of the methylated DBCP analogs to induce renal DNA damage correlated with their necrogenic potential. Experiments using pretreatments that are known to decrease the nephrotoxicity caused by glutathione and cysteine conjugates of several halogenated alkenes were conducted to examine the effect of these pretreatments on DBCP-induced nephrotoxicity. Probenecid, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) and aminooxyacetic acid did not significantly alter renal necrosis or DNA damage induced by DBCP. Based on the absence of any significant isotope effects with the predeutero-DBCP analog, it appears that breaking of a carbon-hydrogen bond is not the rate-limiting step in DBCP-induced nephrotoxicity. Studies with the methylated DBCP analogs indicate that a vicinal dibromo ethyl group must minimally be present for nephrotoxic potential. Furthermore, it seems unlikely that metabolism by renal cysteine conjugate beta-lyase is rate-limiting for DBCP nephrotoxicity.

Role of P-450 activity and glutathione levels in 1,2-dibromo-3-chloropropane tissue distribution, renal necrosis and in vivo DNA damage

Treatments known to alter P-450 activity and glutathione levels were used to elucidate the involvement of P-450 and glutathione S-transferase metabolism in 1,2-dibromo-3-chloropropane (DBCP) organ toxicity in the rat. Phenobarbital pretreatment abolished DBCP-induced renal necrosis, whereas it had only a small effect on initial renal DNA damage. The DBCP levels in plasma and tissues were markedly reduced by phenobarbital pretreatment. Perdeuterated DBCP had much higher plasma and tissue levels than protio-DBCP in phenobarbital-pretreated animals, but perdeuteration was without effect in uninduced animals. This indicates that P-450 metabolism of DBCP is of major importance only in phenobarbital-pretreated animals. In order to study the effects of decreased glutathione levels on renal distribution and toxicity, rats were pretreated with either diethyl maleate or buthionine sulfoximine. The DBCP levels in plasma and tissues showed transitory elevations after diethyl maleate and buthionine sulfoximine pretreatment compared to the control situation. Despite the fact that diethyl maleate and buthionine sulfoximine pretreatments are known to block DBCP-induced DNA damage in vitro, these pretreatments did not significantly alter DBCP-induced renal necrosis nor DNA damage. Thus, a role for glutathione conjugation in DBCP-induced in vivo renal toxicity could not be established in the present study.

Species differences in short term toxicity from inhalation exposure to bromobenzene

Lung, liver and kidney injury were studied in mice, rats and rabbits 48 h after termination of a 4 h inhalation exposure to bromobenzene vapour (250–3400 ppm). Light and electron microscopy of lung tissue revealed injury to Clara cells and adjacent epithelium in mouse bronchioli (bromobenzene concentration 250 ppm and 1000 ppm) and to Clara cells of rat bronchi and bronchioli (1000 ppm bromobenzene) and of rabbit bronchi (2500 ppm and 3400 ppm). Histological and clinicochemical indices of liver damage were found in the same animals, whereas kidney toxicity was observed in mice (two out of ten showed tubular necrosis and elevated concentration of plasma urea) and rats (all had elevated plasma concentrations of creatinine) exposed to 1000 ppm bromobenzene. Inhalation exposure thus produced less kidney injury than expected from previous studies with equimolar doses given intraperitoneally. The mouse was the most severely affected species, followed by the rat, and lastly the rabbit. The animal susceptibility could not be ranked according to the rate of14C-bromobenzene covalent binding in lung or liver, but it was inversely related to the rate of N-demethylation of benzphetamine (indicative of P450IIB activity) in both lung and liver microsomal preparations. Differences in a P450 mediated detoxification could therefore be of importance in species variability to bromobenzene injury.

Formation of reactive 1-nitropyrene metabolites by lung microsomes and isolated lung cells

The metabolism and activation of 1-nitropyrene (1-NP) to reactive intermediates by lung microsomes and isolated lung cells was studied. Mutagenicity of 1-NP metabolites was assayed in Salmonella typhimurium TA98NR, a strain lacking a major component of nitroreductase activity. In the presence of NADPH, microsomes from rabbit, rat and hamster lung metabolized 1-NP to mutagenic products to a similar degree. Pretreatment with a mixture of polychlorinated biphenyls (PCB) decreased the formation of mutagenic metabolites by rabbit lung microsomes, but did not affect the production of mutagens by rat or hamster lung microsomes. 3H-1-NP was metabolized to covalently bound protein products at a rate of 82 and 10 pmol/mg by rabbit and hamster lung microsomes, respectively, whereas no binding was detected in rat lung microsomes. PCB-pretreatment increased covalent protein binding of 3 H-1-NP in lung microsomes from hamster and rat, but decreased the binding in rabbit lung microsomes. High performance liquid chromatography analysis indicated that 3H-1-NP was readily converted to ring-hydroxylated products by rabbit and hamster lung microsomes; the rate was much lower with rat lung microsomes. 3H-1-NP was activated to metabolites that covalently bound to protein in isolated rabbit lung cells, with the following rates being observed: Clara cells > lung digest > type II cells. In contrast, covalent protein binding in cells isolated from rat lung was very low. 1-NP was not activated to products mutagenic for S. typhimurium TA 98 N R when co-incubated with cells isolated either from rabbit or rat lung.

Formation of genotoxic products from N-nitrosoheptamethyleneimine (NHMI), 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone (NNK) and N′-Nitrosonornicotine (NNN) by isolated rabbit lung cells

The genotoxic potentials of N-nitrosoheptamethyleneimine (NHMI), 4-(methylnitrosamino)-1-(3 pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) were studied in fresh preparations of Clara cells and type II cells isolated by centrifugal elutriation and density gradient centrifugation, and macrophages from rabbit lung. The activation of the compounds to bacterial mutagens was assayed in the Salmonella mutagenicity test using strains of TA 100 and TA 1530 preincubated with test chemicals and cells placed in chambers with nucleopore membranes to separate cells and bacteria. Unscheduled DNA synthesis was measured by incorporation of [3H]-thymidine in the cells after exposure to the compounds. NHMI, NNK and NNN were not activated to bacterial mutagens by Clara cells, type II cells or macrophages, presumably because the reactive metabolites generated were not released into the incubation medium. However, NHMI and NNK increased unscheduled DNA synthesis in Clara cells, and the highest repair activity was found after incubation with NNK. The effect of NNN was only marginal. This indicates that NHHI and NNK are genotoxic in the rabbit lung and that the Clara cells are involved in the metabolic activation of these compounds.

Lung and Liver Damage in Mice After Bromobenzene Inhalation: Effects of Enzyme Inducers

AbstractMice were pretreated With the enzyme inducers phenobarbital (PB), polychlorinated biphenyls (PCB) or β-naphthoflavone (BNF), and subsequently exposed to bromoben-zene (BrBz) at 250 and 7000 ppm in inhalation chambers for 4 h. In vivo lung and liver damage were evaluated and compared with results from in vitro covalent binding studies using lung and liver microsomes from mice given the same pretreatments. Reduced lung injury was observed in mice pretreated with PB and PCB compared to controls, whereas BNF pretreatment resulted in decreased liver injury. There were no correlation between the rates of in vitro covalent binding to pulmonary microsomes and the degree of lung injury. However, the slower rate of in vitro binding to liver microsomes compared to controls correlated with the decreased hepatic injury in BNF pretreated mice. An interesting relationship was found in PB- and PCB-pretreated mice between increased rates of covalent binding of bromobenzene to liver microsomes and a decrease in in ...