Evaluation of effects from repeated inhalation exposure of F344 rats to high concentrations of propylene.

Chronic exposure to propylene does not result in any increased incidence of tumors, yet does increase N7-hydroxypropylguanine (N7-HPGua) adducts in tissue DNA. To investigate any potential for genotoxicity (mutagenicity or clastogenicity), male F344 rats were exposed via inhalation to up to 10,000 ppm propylene for 1, 3, or 20 days (6 h/day, 5 days/week). The endpoints examined included gene (Hprt, splenocytes) and chromosomal (bone marrow micronucleus [MN]) mutations, hemoglobin (hydroxypropylvaline, HPVal) adducts in systemic blood, and DNA adducts (N7-HPGua) in several tissues. Similarly exposed female and male F344 rats, implanted with bromodeoxyuridine (BrdU) minipumps, were evaluated for nasal effects (irritation via histopathology and cell proliferation via BrdU). Internal dose measures provided clear evidence for propylene exposure, with HPVal increased for all exposures; N7-HPGua was increased in all tissues from rats exposed for more than 1 day (except lymphocytes). Saturation of propylene conversion to propylene oxide was apparent from the adduct dose-response curves. There were no biologically significant genotoxic effects demonstrated at any exposure level, with no increase in Hprt mutant frequency or in bone marrow MN formation. In addition, no histopathological changes were noted in rodent nasal tissues nor any induction of cell proliferation in nasal tissues. These results demonstrate that repeated exposure of rats to high concentrations of propylene (< or = 10,000 ppm) does not produce evidence of local nasal cavity toxicity or evidence of systemic genotoxicity to hematopoietic tissue, despite the formation of N7-HPGua adducts. In addition, these data indicate that formation of N7-HPGua does not correlate with any measure of genotoxic effect, neither mutagenic nor clastogenic.     

A physiological toxicokinetic model for inhaled propylene oxide in rat and human with special emphasis on the nose.

Chronic exposure to high concentrations of PO induced inflammation in the respiratory nasal mucosa (RNM) of rodents and, for concentrations >or= 300 ppm, caused nasal tumors. Considering the nose to be the most relevant target organ for PO-induced tumorigenicity, we developed a physiological toxicokinetic model for PO in rats and humans. It includes compartments for arterial, venous, and pulmonary blood, liver, muscle, fat, richly perfused tissues, lung, and nose. It simulates inhalation of PO, its distribution into tissues by blood flow, and its elimination by exhalation and metabolism. In nose, lung, and liver of rats, PO conjugation with glutathione (GSH), PO-induced GSH depletion, and formation of PO adducts to DNA are described. Also modeled are PO adducts to hemoglobin of rats and humans. Required partition coefficients and metabolic parameters were derived experimentally or from publications. In rats, simulated PO concentrations in blood and GSH levels in tissues agreed with measured data. If compared with reported values, levels of adducts with hemoglobin were underpredicted up to a factor of about 2. Adducts with DNA differed up to a factor of 3. Hemoglobin adducts predicted for PO-exposed workers were 1.5-1.9 times higher than the reported ones. Considering identical conditions of PO exposure, similar PO concentrations in RNM were modeled for rats and humans. Also, PO concentrations in blood, about 1/30th of those in RNM, were similar in both species. Since the model was evaluated on all available data in rats and humans, we consider it to be useful for estimating the risk from inhalation exposure to PO.     

DNA methylation, cell proliferation, and histopathology in rats following repeated inhalation exposure to dimethyl sulfate

Dimethyl sulfate (DMS) is an alkylating agent that is carcinogenic to the respiratory tract of rodents. DNA adducts, cell proliferation, and histopathology were assessed in rats to better understand the molecular dosimetry and tissue dynamics associated with repeated inhalation exposure to DMS. For DNA methylation, rats were exposed to DMS vapor 6 h/day for up to 10 days to 0.0, 0.1, 0.7 and 1.5 ppm. N7-Methylguanine and N3-methyladenine were detected in neutral thermal hydrolysates of DNA isolated from respiratory tract tissues by high-performance liquid chromatography (HPLC) using fluorescence and ultraviolet (UV) detection. DNA methylation was greatest in DNA isolated from nasal respiratory mucosa, less in olfactory, and little was found in lung. N7-Methylguanine levels in respiratory mucosa approached steady-state levels by day 5, and N7-methylguanine persistence following exposure for 5 consecutive days was also determined. Loss of N7-methylguanine from respiratory and olfactory mucosa appeared to follow first-order kinetics. N3-Methyladenine levels were at or below detection limits in all samples. The effect of DMS on histopathology and cell proliferation in the nasal epithelium was also investigated. Rats were exposed nose-only for 2 wk to DMS vapor at concentrations of 0, 0.1, 0.7, or 1.5 ppm. Inhalation exposure to DMS induced degenerative and inflammatory changes in nasal epithelium at >or=0.7 ppm. Cell proliferation evaluations showed a trend towards an increased response at 1.5 ppm. These experiments demonstrate that DMS can induce cytotoxic and proliferative effects and is a potent methylating agent of the nasal mucosa in vivo. These experiments will provide data for the development of dosimetry models useful for risk extrapolation.     

Effects of propylene oxide exposure on rat nasal respiratory cell proliferation.

Long-term exposure of rodents to propylene oxide (PO) induced inflammation, respiratory cell hyperplasia, and nasal tumors at concentrations >/= 300 ppm, suggesting a possible role for cytotoxicity and compensatory cell proliferation in PO carcinogenesis. In this study, the effects of PO exposure on histopathology and cell proliferation in nasal and hepatic tissues were studied in male F344 rats exposed by inhalation for 3 or 20 days (0, 5, 25, 50, 300, and 500 ppm). Histopathology revealed an increase in mucous cell hyperplasia in the anterior nasal passages after 20 days of exposure (>/=300 ppm). This was associated with the formation of goblet cell nests. Cell proliferation was measured in the respiratory epithelium (NRE; mucociliary and transitional) lining the anterior nasal passages, the nasopharyngeal meatus (NPM), and the liver using BrdU administered with 3-day osmotic pumps. Significant increases in cell proliferation occurred (>3.6-fold) in the mucociliary epithelium lining the anterior nasal cavity at and above 300 ppm for both exposure periods. In the mucociliary epithelium, the 20-day labeling was commonly associated with nests of goblet cells. Significant increases in cell proliferation (>2.3-fold) were observed in the transitional epithelium at 500 ppm after 3 days of exposure and at 300 and 500 ppm after 20 days of exposure. Significant increases in cell proliferation in the NPM (>2.8-fold) were evident at 500 ppm PO after 3 days and at 300 and 500 ppm PO after 20 days of exposure. No exposure-related changes in cell proliferation were observed in the liver. These studies demonstrate a clear concordance between the site and exposure concentration for tumor induction and those causing significant increases in cell proliferation in the rat nose.     

Proliferative responses of rat nasal epithelia to ozone

The epithelium of the female Fischer 344/N rat anterior nasal cavity was examined and found to be composed of four types of epithelia: squamous, ciliated respiratory, nonciliated cuboidal/transitional, and olfactory. DNA replication in these tissues was monitored by bromodeoxyuridine (BrdUrd) incorporation. Labeled cells were identified using a monoclonal antibody recognizing BrdUrd. Ciliated respiratory, nonciliated cuboidal/transitional, and olfactory epithelia from control animals had a low level of DNA replication (1 labeled cell/mm basal lamina); in contrast, the squamous epithelium contained 40 labeled cells per millimeter basal lamina. Female Fischer 344/N rats were exposed to 0.0, 0.12, 0.27, or 0.8 ppm ozone, 6 hr/day for up to 7 days. Observations were made after 3 or 7 days of exposure and after 3 or 7 days of recovery from the 7-day exposure. Following exposure to 0.8 ppm ozone, a transient but marked increase in DNA replication was seen in the nonciliated cuboidal/transitional, while in ciliated respiratory and olfactory epithelia the transient increase in DNA replication was less marked. This increase was prominent after 3 days of exposure and absent by 7 days of exposure in all but the cuboidal/transitional epithelium. Exposure to 0.8 ppm ozone for either 3 or 7 days resulted in hyperplasia of the cuboidal epithelium. A depressed level of DNA replication was seen in the squamous epithelium following 7 days of recovery from 7 days of ozone exposure to 0.8 ppm ozone. This study shows that there are regional differences in DNA replication within the anterior nasal epithelium of the rat and that these levels are modulated by exposure to irritants. The cuboidal/transitional epithelium was the most responsive epithelial cell type to the effects of ozone exposure and may, therefore, provide a sensitive indicator of irritant damage to the respiratory tract.     

DNA Methylation,Cell Proliferation,and Histopathology in Rats Following Repeated Inhalation Exposure to Dimethyl Sulfate

Dimethyl sulfate (DMS) is an alkylating agent that is carcinogenic to the respiratory tract of rodents. DNA adducts, cell proliferation, and histopathology were assessed in rats to better understand the molecular dosimetry and tissue dynamics associated with repeated inhalation exposure to DMS. For DNA methylation, rats were exposed to DMS vapor 6 h/day for up to 10 days to 0.0, 0.1, 0.7 and 1.5 ppm. N⁷-Methylguanine and N³-methyladenine were detected in neutral thermal hydrolysates of DNA isolated from respiratory tract tissues by high-performance liquid chromatography (HPLC) using fluorescence and ultraviolet (UV) detection. DNA methylation was greatest in DNA isolated from nasal respiratory mucosa, less in olfactory, and little was found in lung. N⁷-Methylguanine levels in respiratory mucosa approached steady-state levels by day 5, and N⁷-methylguanine persistence following exposure for 5 consecutive days was also determined. Loss of N⁷-methylguanine from respiratory and olfactory mucosa appeared to follow first-order kinetics. N³-Methyladenine levels were at or below detection limits in all samples. The effect of DMS on histopathology and cell proliferation in the nasal epithelium was also investigated. Rats were exposed nose-only for 2 wk to DMS vapor at concentrations of 0, 0.1, 0.7, or 1.5 ppm. Inhalation exposure to DMS induced degenerative and inflammatory changes in nasal epithelium at ≥0.7 ppm. Cell proliferation evaluations showed a trend towards an increased response at 1.5 ppm. These experiments demonstrate that DMS can induce cytotoxic and proliferative effects and is a potent methylating agent of the nasal mucosa in vivo. These experiments will provide data for the development of dosimetry models useful for risk extrapolation.     

Effects of intermittent exposure to aflatoxin B1 on DNA and RNA adduct formation in rat liver: dose-response and temporal patterns.

We studied the effects of intermittent exposure to aflatoxin B1 (AFB1) on hepatic DNA and RNA adduct formation. Fisher-344 male rats were fed 0.01, 0.04, 0.4, or 1.6 ppm of AFB1 intermittently for 8, 12, 16, and 20 weeks, alternating with 4 weeks of dosing and 4 weeks of rest. Other groups of rats were fed 1.6 ppm of AFB1 continuously for 4, 8, 12, and 16 weeks. Control rats received AFB1-free NIH-31 meal diet. AFB1-DNA and -RNA adducts were measured by HPLC with fluorescence detection. The data are presented as total DNA or RNA adducts. The DNA and RNA adduct levels increased or decreased depending on the cycles of dosing and rest. Rats removed from treatment 1 month after 1 or 2 dosing cycles (8 and 16 weeks of intermittent exposure) showed approximately a twofold decrease in DNA adduct levels and a two- to elevenfold decrease in RNA adduct levels compared with rats euthanized immediately after the last dosing cycle (12 and 20 weeks of intermittent exposure). Our data indicate that DNA and RNA adducts increased linearly, from 0.01 ppm to 1.6 ppm of AFB1 after 12 and 20 weeks of intermittent treatment. A linear dose response was also apparent for DNA but not for RNA adducts after 8 and 16 weeks of treatment. As biomarkers of exposure, AFB1-RNA adducts were three to nine times more sensitive than AFB1-DNA adducts but showed greater variability. These results suggest that binding of AFB1 to hepatic DNA is a linear function of the dose, regardless of the way this is administered. The dose-response relationship for RNA adducts depends on the length of the no-dosing cycles and on the turnover rate of RNA.     

Hemoglobin and DNA adduct formation in Fischer-344 rats exposed to 2,4- and 2,6-toluene diamine

 Using gas chromatography/mass spectrometry for detection of hemoglobin adducts, and ³²P-postlabelling for DNA adducts, we examined macromolecular binding in Fischer-344 rats administered 2,4- or 2,6-toluene diamine (TDA). The dose-response and correlative relationship between the two macromolecules were investigated over a range of doses (0–250 mg/kg). The time course of adduct formation and removal was also examined. Both TDA isomers induced formation of hemoglobin adducts, but only the 2,4-isomer induced DNA binding. Maximum hemoglobin and DNA adduct levels were detected 24 h following administration. Both hemoglobin and DNA binding increased in a dose-dependent manner. Hemoglobin adduct clearance demonstrated a nonlinear decay, with adduct loss occurring faster than normal erythrocyte clearance. The effects of metabolic inhibitors on adduct formation were examined using piperonyl butoxide and pentachlorophenol to inhibit p450 isozymes and sulfotransferase, respectively. Microsomal enzymatic activation was critical to hemoglobin adduct formation with inhibition by piperonyl butoxide reducing adduct yields by over 90%. Sulfation did not appear to play a significant role in TDA-induced hemoglobin adduct formation. Received: 19 July 1995/Accepted: 9 January 1996     

Two-day inhalation toxicity study of methyl iodide in the rat

The effects of inhaled methyl iodide (MeI) on clinical pathology parameters, glutathione (GSH) tissue levels, serum thyroid hormone and inorganic iodide concentrations, S-methylcysteine hemoglobin concentrations, and liver UDP-glucuronyltransferase activity were studied in the rat. Male rats were exposed by whole-body inhalation to 0, 25, or 100 ppm MeI, 6 h/day for up to 2 days. Serum cholesterol concentrations (both high-density lipoprotein [HDL] and low-density lipoprotein [LDL] fractions) were increased and triglycerides were decreased at both exposure levels. Serum thyroid-stimulating hormone (TSH) concentrations were increased at 25 and 100 ppm, and serum triiodothyronine (T₃) and thyroxine (T₄) concentrations were decreased at 100 ppm. There was no change in either reverse triiodothyronine (rT₃) or UDP-glucuronyltransferase activity at either exposure level. A dose- and time-dependent reduction in GSH levels in blood, kidney, liver, and nasal tissue was observed, with the greatest reduction in nasal tissue (olfactory and respiratory epithelium). MeI exposure also resulted in a substantial dose- and time-dependent increase in both serum inorganic iodide and red blood cell S-methylcysteine hemoglobin adducts. These results indicate that following inhalation exposure, MeI is rapidly metabolized in blood and tissue of rats, resulting in methylation products and release of inorganic iodide.     

Interspecies dose extrapolation for inhaled dimethyl sulfate: a PBPK model-based analysis using nasal cavity N7-methylguanine adducts

Dimethyl sulfate (DMS) is a volatile sulfuric acid ester used principally as a methylating agent in a wide variety of industrial applications. DMS reacts with organic macromolecules by a SN2 mechanism. The weight of experimental evidence suggests that DMS possesses genotoxic and carcinogenic potential. Inhalation studies have shown that repeated exposure to DMS leads to tumors in the nasal cavity and lower respiratory tract in both rats and mice. Here we present a quantitative assessment for cross-species dose extrapolation for inhaled DMS using a physiologically based pharmacokinetic (PBPK) model. The model is designed to simulate N7-methylguanine (N7 mG) DNA adduct levels in the nasal mucosa following DMS exposure in rats and humans. This model was parameterized and predictions were tested by comparison against experimentally measured N7 mG DNA adduct levels in rat nasal mucosa following inhalation exposure to DMS. The model-based interspecies dose comparison, using N7 mG adduct levels in the nasal respiratory tissue as the appropriate dose metrics, predicts a dose rate seven times higher in rats compared to humans.     

Dimethyl Sulfate Uptake and Methylation of DNA in Rat Respiratory Tissues Following Acute Inhalation

Adult male CrlCD:BR rats were exposed nose-only to several concentrationsof dimethyl sulfate (DMS) vapors to determine the relationshipsbetween vapor uptake and DNA methylation. Following DMS exposure,nasal respiratory and olfactory mucosa and lung tissue wereremoved and DNA was isolated for the analysis of methylatedpurines. DMS vapor uptake was complex and related to exposureconcentration; clearance appeared to increase with increasingDMS concentrations between 0.5 and 8 ppm. Plethysmorgraphicmeasurements correlated with the time-dependent disappearanceof dimethyl sulfate from a closed exposure apparatus. Abovean initial DMS concentration of 8 ppm, sensory irritancy apparentlyaltered normal respiratory parameters, clearance, and regionalDNA methylation. DMS-dependent N7-methylguanine formation inDNA isolated from nasal respiratory mucosa was detectable 30mm following a 20-min exposure to an initial DMS concentrationof 1 ppm. DMS-dependent methylation of DNA, as evidenced byN7-methylguanine and N3-methyladenine formation, showed concentration-responserelationships in all tissues examined and was correlated withvapor uptake. DNA adduct formation showed regional differencescharacteristic of the absorption of a water-soluble vapor; methylationwas greatest in DNA isolated from respiratory mucosa, less inolfactory, and little in lung. Repair of N7-methylguanine didnot appear to be significantly different between nasal respiratoryand olfactory tissues. These experiments demonstrate that DMSis a potent methylating agent of nasal mucosa in vivo.     

Determination of N2-hydroxymethyl-dG adducts in the nasal epithelium and bone marrow of nonhuman primates following 13CD2-formaldehyde inhalation exposure

The presence of endogenous and exogenous N(2)-hydroxymethyl-dG adducts in DNA from the nasal mucosa and bone marrow of cynomolgus macaques exposed to 1.9 and 6.1 ppm of [(13)CD(2)]-formaldehyde for 6 h a day for 2 consecutive days was investigated using a highly sensitive nano-UPLC-MS/MS method with a limit of detection of 20 amol. Both exogenous and endogenous adducts were readily detected and quantified in the nasal tissues of both exposure groups, with an exposure dependent increase in exogenous adducts observed. In contrast, only endogenous adducts were detectable in the bone marrow, even though ～10 times more DNA was analyzed.     

Interspecies Dose Extrapolation for Inhaled Dimethyl Sulfate: A PBPK Model-Based Analysis using Nasal Cavity N7-Methylguanine Adducts

Dimethyl sulfate (DMS) is a volatile sulfuric acid ester used principally as a methylating agent in a wide variety of industrial applications. DMS reacts with organic macromolecules by a S_N2 mechanism. The weight of experimental evidence suggests that DMS possesses genotoxic and carcinogenic potential. Inhalation studies have shown that repeated exposure to DMS leads to tumors in the nasal cavity and lower respiratory tract in both rats and mice. Here we present a quantitative assessment for cross-species dose extrapolation for inhaled DMS using a physiologically based pharmacokinetic (PBPK) model. The model is designed to simulate N⁷-methylguanine (N⁷mG) DNA adduct levels in the nasal mucosa following DMS exposure in rats and humans. This model was parameterized and predictions were tested by comparison against experimentally measured N⁷mG DNA adduct levels in rat nasal mucosa following inhalation exposure to DMS. The model-based interspecies dose comparison, using N⁷mG adduct levels in the nasal respiratory tissue as the appropriate dose metrics, predicts a dose rate seven times higher in rats compared to humans.     

DNA adducts in liver and leukocytes of flounder (Platichthys flesus) experimentally exposed to benzo

In the present study the levels of hydrophobic DNA adducts detected by 32P-postlabelling were followed in liver and leukocytes of flounder (Platichthys flesus) over 10 days following single i.p. injections of two doses of BaP (10 and 50 mg kg(-1) fish weight, respectively). DNA adducts were detected in both tissues of exposed fish 2 days post injection and continued to rise on day 5 and day 10. In flounder exposed to the lower dose of BaP, the levels of hepatic DNA adducts reached higher values on the fifth day compared with flounder exposed to the higher dose. However, at the end of the experiment, the DNA adduct level was again higher in fish from the high dose group compared with the low dose group. There was no substantial increase of DNA adducts in liver of flounder from the low dose group after day 5, while the adduct levels in flounder liver from the high dose group increased throughout the experiment. Earlier studies detecting DNA adducts in BaP-exposed flatfish with the 32P-postlabelling technique have reported declining adduct levels from about 2 days after the exposure, regardless of exposure route. In contrast, the results from our study did not confirm a rapid increase and successive decline of hydrophobic adducts in liver of BaP-exposed flounder.     

DNA--Carcinogen Adducts in Circulating Leukocytes as Indicators of Arylamine Carcinogen Exposure

DNA—carcinogen adducts in leukocytes and putative targettissues (liver and urinary bladder) of C57BL/6J mice were measuredby ³²P-postlabeling and HPLC analysis after controlled exposureto the arylamine carcinogen 2-aminofluorene (2-AF). After anacute exposure via ip injection, adducts were detected at 3hr in leukocytes, liver, and bladder. The disappearance of DNA-carcinogenadducts in liver and leukocytes were parallel over the 24-hrperiod studied. Following a 7-day continuous exposure to 2-AFvia drinking water, adduct levels in leukocytes and target tissueswere responsive to dose at 30, 100, and 300 ppm. Adduct levelsat the highest dose reached 17,000 fmol/mg DNA in leukocytes,1900 fmol/mg in liver, and 2300 fmol/mg in bladder. Althoughadduct levels after 7 days were highest in leukocytes, adductswere not detectable in leukocytes 7 days after discontinuingexposure. In contrast, liver and bladder retained approximately50 and 75% of their respective adduct levels 7 days after exposurewas stopped. The results indicate that circulating leukocytesmay be useful as indicators of current exposure to arylaminecarcinogens. Circulating leukocytes may also be useful as biologicalmonitors of DNA damage in arylamine target tissues during chronicexposure to these compounds. Some important differences in persistenceof DNA-carcinogen adducts between leukocytes and target tissueswere observed.     

Sustained overexpression of CYP1A1 and 1B1 and steady accumulation of DNA adducts by low-dose, continuous exposure to benzo[a]pyrene by polymeric implants

Many carcinogenesis and tumorigenesis studies reported in the past several decades have relied upon bolus dose(s) of test compounds to determine their DNA damage and carcinogenic potential. The high doses are far from the human scenario where exposure is almost always to low doses and for long duration. In this study, we report a novel polymeric implant system that provides continuous ("24/7") exposure to low doses using benzo[a]pyrene (BP) as a model carcinogen. Cylindrical implants (1 cm length, 3.2 mm diameter; 10 mg BP/100 mg implant) prepared from polycaprolactone:F68 (9:1) showed controlled release in vitro for long duration. To determine the rate of release and biochemical effects in vivo, groups of female Sprague-Dawley rats received either no treatment or subcutaneous sham or BP implants (1 cm, 10% load) and were euthanized after 6, 15, 30, and 180 days; the average dose of BP by the implant route was 16.7 ± 3 μg/rat. For comparison, rats were also treated with a single bolus dose of BP intraperitoneally (10 mg/rat) and euthanized at 6, 15, and 30 days. DNA adducts analyzed by (32)P-postlabeling in the lung and liver increased steadily with time with levels reaching 31 ± 3 and 17 ± 6 adducts/10(9) nucleotides, respectively, after 25 weeks; the adduct burden in the mammary tissue initially increased but then declined with time presumably due to high cell turn over. In contrast, the bolus dose treatment showed the highest DNA adduct levels after 6 days, followed by a steady decline. The steady accumulation of tissue DNA adducts in the implant groups corroborates the sustained overexpression of CYP1A1 and 1B1, the cytochrome P450s involved in the conversion of BP to its electrophilic metabolites. In contrast, the overexpression of CYP1A1 and 1B1 resulting from the bolus dose of BP lasted only for a few days. This is the first demonstration revealing that low-dose, continuous exposure to environmental polycyclic aromatic hydrocarbons such as BP can render sustained expression of CYPs and steady accumulation of tissue DNA adducts. On the basis of our recent study in which we showed the presence of 17β-estradiol in the lung, the sustained overexpression of CYP1A1 and 1B1 due to continuous exposure to BP may increase the susceptibility to estrogen-mediated carcinogenicity.     

Mutagenicity and DNA adduct formation by the urban air pollutant 2-nitrobenzanthrone.

2-Nitrobenzanthrone (2-NBA) has recently been detected in ambient air particulate matter. Its isomer 3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust. The highest mutagenic activity of 2-NBA tested in Salmonella typhimurium was exhibited in strain TA1538-hSULT1A1 expressing human sulfotransferase (SULT) 1A1. 2-NBA also induced mutations in Chinese hamster lung V79 cells expressing human N-acetyltransferase 2 or SULT1A1, but no mutagenicity was observed in the parental cell line. DNA adduct formation in vitro was examined in different human cell lines by thin-layer chromatography (32)P-postlabeling. Whereas 3-NBA formed characteristic DNA adducts in lung A549, liver HepG2, colon HCT116, and breast MCF-7 cells, 2-NBA-derived DNA adducts were only observed in A549 and HepG2 cells, indicating differences in the bioactivation of each isomer. The pattern of 2-NBA-derived DNA adducts in both cell lines consisted of a cluster of up to five adducts. In HepG2 cells DNA binding by 2-NBA was up to 14-fold lower than by 3-NBA. DNA adduct formation of 2-NBA was also investigated in vivo in Wistar rats treated with a single dose of 2, 10, or 100 mg/kg body weight (bw). No DNA adduct formation was detected at doses of up to 10 mg/kg bw 2-NBA, even though 3-NBA induced DNA adducts at a dose of 2 mg/kg bw. Only after administration of one high dose of 100 mg/kg bw 2-NBA was a low level of DNA adduct formation detected, and then only in lung tissue. Density functional theory calculations for both NBAs revealed that the nitrenium ion of the 3-isomer is considerably more stable ( approximately 10 kcal/mol) than that of the 2-isomer, providing a possible explanation for the large differences in DNA adduct formation and mutagenicity between 2- and 3-NBA.     

Stability of hemoglobin and albumin adducts of naphthalene oxide, 1,2-naphthoquinone, and 1,4-naphthoquinone.

Naphthalene is an important industrial chemical, which has recently been shown to cause tumors of the respiratory tract in rodents. It is thought that one or more reactive metabolites of naphthalene, namely, naphthalene-1,2-oxide (NPO), 1,2-naphthoquinone (1,2-NPQ), and 1,4-naphthoquinone (1,4-NPQ) contribute to the tumorigenicity of this chemical. These electrophiles are all capable of covalent binding to macromolecules including DNA and proteins. The stability of cysteinyl adducts of NPO, 1,2-NPQ, and 1,4-NPQ were investigated in both hemoglobin (Hb) and albumin (Alb) of male F344 rats following a single administration of 2 different doses (400 or 800 mg naphthalene per kg body weight). To assess the stability of Alb adducts, we compared the rates of NPO-Alb turnover (half-life of approximately 2 days) and 1,2-NPQ-Alb (half-life of approximately 1 day) to the normal turnover rate of Alb in the rat (half-life = 2.5-3 days). Based on the rapid turnover of these adducts relative to Alb itself, we concluded that they were unstable. However, the stability of Alb adducts was not affected by the dose of naphthalene administered (400 or 800 mg/kg). In contrast, NPO-Hb adducts were relatively stable (rate constant of adduct instability 相似文献   

Glutathione depletion is a major determinant of inhaled naphthalene respiratory toxicity and naphthalene metabolism in mice.

Naphthalene (NA) is metabolized to highly reactive intermediates that are primarily detoxified by conjugation to glutathione (GSH). Intraperitoneal administration of naphthalene causes substantial loss of both hepatic and respiratory GSH, yet only respiratory tissues are injured in mice. The liver supplies GSH to other organs via the circulation, making it unclear whether respiratory GSH losses reflect in situ respiratory depletion or decreased hepatic supply. To address this concern, mice were exposed to naphthalene by inhalation (1.5-15 ppm; 2-4 h), thereby bypassing first-pass hepatic involvement. GSH levels and histopathology were monitored during the first 24 h after exposure. Half of the mice were given the GSH depletor diethylmaleate (DEM) 1 hour before naphthalene exposure. Lung and nasal GSH levels rapidly decreased (50-90%) in mice exposed to 15 ppm naphthalene, with cell necrosis throughout the respiratory tract becoming evident several hours later. Conversely, 1.5 ppm naphthalene caused moderate GSH loss and only injured the nasal olfactory epithelium. Neither naphthalene concentration depleted hepatic GSH. Animals pretreated with DEM showed significant GSH loss and injury in nasal and intrapulmonary airway epithelium at both naphthalene concentrations. DEM treatment, perhaps by causing significant GSH loss, decreased water-soluble naphthalene metabolite formation by 48% yet increased NA-protein adducts 193%. We conclude that (1) GSH depletion occurs in airways independent of hepatic function; (2) sufficient GSH is not supplied by the liver to maintain respiratory GSH pools, or to prevent injury from inhaled naphthalene; and (3) GSH loss precedes injury and increases protein adduct formation.     

Distribution of DNA adducts in the respiratory tract of rats exposed to diesel exhaust

Diesel exhaust, inhaled chronically at high concentrations, was previously found to be a pulmonary carcinogen in rats. The exhaust-induced tumors were located exclusively in the peripheral lung, although all of the respiratory tract tissues were exposed to the exhaust. The purpose of this study was to determine whether there were differences in the level of DNA adducts among the regions of the respiratory tract that paralleled the site of tumors. Groups of male F344/N rats were exposed 7 hr/day, 5 day/week for 12 weeks to diesel engine exhaust at a soot concentration of 10 mg/m3 or were sham-exposed to air. The maxilloturbinates, ethmoturbinates, trachea, left mainstem bronchus (airway generation 1), axial airway (airway generations 2-12), and peripheral lung tissue were dissected from the respiratory tract. DNA was isolated from the dissected samples and analyzed for the presence of adducts using the 32P-postlabeling assay. Chromatographic maps of DNA adducts demonstrated unique patterns of DNA adducts for each of the regions. The highest level of total DNA adducts occurred in peripheral lung tissue (approximately 20 adducts per 10(9) bases). The level of DNA adducts detected in the nasal tissues was about one-fourth to one-fifth that detected in peripheral lung. There were less than three adducts per 10(9) bases in each of the regions of the major conducting airways (i.e., trachea, bronchi, axial airway). In control rats, levels of DNA adducts ranged from one adduct per 10(9) bases (mainstem bronchi, axial airway) to about nine adducts per 10(9) bases (parenchyma). The data from this study indicate that higher levels of total DNA adducts and exhaust-induced adducts (i.e., exposed minus control adducts) were present in tissues where exhaust-induced tumors were located. These data suggest that DNA adduct levels in discrete locations of the respiratory tract may be good measures of the "effective dose" of carcinogenic compounds.