Comparative QSAR evidence for a free-radical mechanism of phenol-induced toxicity.

Phenol and 14 substituted-phenols were tested for their ability to impair epithelial cell membrane integrity in WB rat liver cells as determined by an increase in lactate dehydrogenase release. Two quantitative structure-activity relationship (QSAR) regression equations were developed which showed that separate mechanisms of phenolic cytotoxicity are important - nonspecific toxicity due to hydrophobicity and formation of phenoxyl radicals. The equations most predictive of phenol toxicity are denoted as log1/C=-0. 98sigma(+)+0.77logP+0.23 or log1/C=-0.11BDE+0.76logP+0.21, respectively, where C is the minimum concentration of substituted-phenol required for a toxic response. P is the octanol-water partition coefficient, sigma(+) is the electronic Hammett parameter and BDE is the OH homolytic bond dissociation energy. In the literature, phenol toxicity correlated to sigma(+) is rare, but there is strong evidence that phenols possessing electron-releasing groups may be converted to toxic phenoxyl radicals. A common feature in a variety of cells is generation of elevated amounts of reactive oxygen species (ROS) associated with a rapid growth rate. The slightly elevated cancer risk associated with the use of Premarin may be due to phenoxyl-type radicals derived from one or more of its components.

Human urine mutagenicity study comparing cigarettes which burn or primarily heat tobacco.

Cigarette smokers have been reported to void urine which is more mutagenic, as measured in the Ames assay, than urine voided by non-smokers. Condensate from the mainstream smoke of a cigarette which primarily heats tobacco (test cigarette) has shown significantly reduced mutagenicity in a battery of in vitro genotoxicity assays compared with tobacco-burning cigarettes. The objective of this study was to determine whether the reduction in mutagenic activity observed in the in vitro assays would be reflected in the urine of smokers of the test cigarette. Twenty smokers were enrolled in a 4-week crossover study, with each smoker consuming test cigarettes ad libitum for a week and their usual brand of tobacco-burning cigarettes the other 3 weeks. Diet was strictly controlled throughout the study, and broiled and pan-fried meat was not served to minimize ingestion of mutagenic protein pyrolysis products. There was no statistically significant difference (p = 0.06) in consumption of tobacco-heating and tobacco-burning cigarettes. There were no statistically significant differences (p = 0.22) in salivary cotinine concentrations for smokers when smoking either tobacco-burning or tobacco-heating cigarettes. Urinary nicotine (ng/mg creatinine) was not different (p = 0.31) for smokers when smoking either tobacco-burning or tobacco-heating cigarettes. Urinary cotinine (ng/mg creatinine) was 32% lower (p = 0.0004) when smoking tobacco-heating cigarettes as compared with smoking tobacco-burning cigarettes. Twenty-four-hour urine samples were collected twice weekly, concentrated using XAD-2 resin and tested in Ames strains TA98 and YG1024 with metabolic activation. Tobacco-burning cigarette smokers experienced a 79% reduction in urinary mutagenicity as measured in strain YG1024 and a 72% reduction as measured in strain TA98 during the week that they smoked the tobacco-heating cigarette while maintaining a fixed dietary regimen. The results of this study indicate that smokers of tobacco-heating cigarettes void urine which is significantly less mutagenic than urine voided by smokers of tobacco-burning cigarettes.

Human urine mutagenicity study comparing cigarettes which burn or only heat tobacco.

Cigarette smokers have been reported to void urine which is more mutagenic, as measured in the Ames bacterial mutation assay, than urine voided by non-smokers. Condensate from the mainstream smoke of a cigarette which heats, but does not burn tobacco (test cigarette) showed no evidence of mutagenicity in a battery of in vitro genotoxicity assays under conditions in which condensate from the mainstream smoke of cigarettes that burn tobacco was mutagenic. The objective of this study was to determine whether the absence of mutagenic activity observed in the in vitro assays would be reflected in the urine of smokers of the test cigarette. 72 subjects (31 smokers and 41 non-smokers) were enrolled in a 6-week study, with the smokers randomly divided into 2 groups. The study was designed as a double crossover, with each smoker smoking both test (tobacco-heating) and reference (tobacco-burning) cigarettes. This design allowed each smoker to serve as his or her own control while at the same time allowing comparisons between groups of non-smokers and smokers of both test and reference cigarettes. 24-h urine samples were collected twice a week and concentrated using XAD-2 resin. Urine concentrates were tested in Ames bacterial strains TA98 and TA100, with and without metabolic activation and with and without beta-glucuronidase/aryl sulfatase. Individuals who smoked the test cigarette voided urine which was significantly less mutagenic than that voided when they smoked reference cigarettes. The mutagenicity of urine from smokers who smoked the test cigarette and non-smokers did not differ under any of the assay conditions used in this study.

Analysis of cytogenetic effects in bone-marrow cells of rats subchronically exposed to smoke from cigarettes which burn or only heat tobacco.

The genotoxic effects of 90-day nose-only exposures to smoke from new cigarettes, which heat but do not burn tobacco (New), or from reference cigarettes, which burn tobacco, were evaluated in Sprague-Dawley rats by examining the cytogenetic endpoints of sister-chromatid exchanges (SCE), chromosome aberrations, and micronuclei in bone-marrow cells. The concentrations of wet total particulate matter (WTPM) and carbon monoxide in the smoke from both cigarette types were similar. The mainstream smoke from both New and reference cigarettes was adjusted to WTPM concentrations of approx. 200 and 400 micrograms/l for low and high smoke exposure. Rats were exposed to smoke 1 h per day, 5 days per week for 13 consecutive weeks. Inhalation of smoke by the exposed animals was confirmed by analysis of blood carboxyhemoglobin and plasma nicotine. Examination of bone-marrow cells following the final day of exposure showed that smoke from neither the New nor reference cigarette induced a positive response in the SCE, chromosome aberration, or micronucleus assays in rats.

Gap junction intercellular communication and cytotoxicity in normal human cells after exposure to smoke condensates from cigarettes that burn or primarily heat tobacco.

Heating tobacco, rather than burning it, reduces tobacco combustion and pyrolysis products. This study tested the hypothesis that the simplified smoke chemistry of a cigarette which primarily heats tobacco (TOB-HT) significantly reduces the potential to alter the structure or function of cellular plasma membranes relative to low "tar" 1R4F and ultra low "tar" lR5F Kentucky reference cigarettes which burn tobacco. Gap junction intercellular communication (GJIC) and lactate dehydrogenase release (LDH) were used to quantify functional and structural changes to the plasma membrane, respectively. Cigarette smoke condensate (CSC) from the mainstream smoke of TOB-HT, lR4F and 1R5F cigarettes were compared in the GJIC and LDH release assays following a 1-hr exposure in vitro. Human bronchial/tracheal epithelial cells, coronary artery endothelial cells, coronary artery smooth muscle cells, foreskin keratinocytes and the WB-344 rat liver epithelial cell line were studied. TOB-HT did not inhibit GJIC in any of the human cell types tested (P0.05) at concentrations where 1R4F and lR5F did inhibit GJIC (P<0.05). TOB-HT did not elevate LDH release (P0.05) when tested at concentrations where lR4F and lR5F did elevate LDH release (P<0.05). Our results suggest that CSC from TOB-HT cigarettes is less damaging to the structure or function of the cellular plasma membranes of a variety of human cell lines than CSC from 1R4F and 1R5F tobacco burning reference cigarettes.

Molecular toxicology endpoints in rodent inhalation studies.

Although histopathology will continue to be essential for assessing the results of rodent inhalation studies, molecular toxicology endpoints are of increasing importance, as these techniques often complement and extend histopathological examinations. One of the primary uses of molecular toxicology is determining the delivered dose of the inhaled material to macromolecules in target tissues. During inhalation studies this is most often done by measuring DNA adducts in the respiratory tract. DNA adducts may be measured specifically (e.g. using monoclonal antibodies or mass spectrometry) or non-specifically (e.g. by using the 32P-post-labeling assay). Another major use of molecular toxicology techniques is the assessment of cellular and molecular changes in target tissues which may precede or be more sensitive than histopathologic alterations. For example, rates of cellular DNA synthesis occurring in target tissues may be quantified at any time during the study by administering the animals either radiolabelled thymidine or the non-radiolabelled thymidine analog bromodeoxyuridine (BrdU). Pulmonary changes may be assessed in bronchoalveolar lavage fluid using either cellular (e.g. macrophage number, granulocyte number) or biochemical (e.g. alkaline phosphatase, lactate dehydrogenase) techniques. The potential of the inhaled material to produce genetic alterations may be evaluated by examining the chromosomes of pulmonary alveolar macrophages for cytogenetic changes. To illustrate the use of these endpoints, an experiment was conducted to determine the molecular toxicology of aged and diluted sidestream smoke (a surrogate for environmental tobacco smoke) in rodent inhalation studies. The endpoints measured were DNA adducts in target and non-target tissue, chromosome aberrations in pulmonary alveolar macrophages, and DNA synthesis in the epithelial lining of the nasal turbinates.

Correlation of hematologic markers of inflammation and lung function: a comparison of asymptomatic smokers and nonsmokers.

Increased inflammation of the peripheral airways has been implicated as a cause of pulmonary function impairment. However, little information is available on the correlation between subclinical decrements of pulmonary function and inflammation in asymptomatic individuals. A relationship between markers of inflammation and lung function may be useful in predicting the early onset of lung function impairment. The purpose of this study was to investigate the correlation of hematologic markers of inflammation and spirometry in asymptomatic smokers and nonsmokers. The specific objectives of this study were twofold. The first objective was to quantify and compare the spirometric measures of lung function in smokers and nonsmokers having similar demographic and lifestyle characteristics. The second objective was to define the correlation between these spirometric measurements and hematologic markers of inflammation (white blood cells, monocytes, basophils, PGE1, IgG, and IgE). Systemic blood samples and spirometric measurements were obtained from 61 age-matched (33 +/- 9 years) healthy, asymptomatic smokers and nonsmokers, with similar self reported lifestyles (i.e., food, alcohol, vitamin consumption and exercise). Both male and female smokers self reported a higher coffee consumption (P < 0.05) compared to nonsmokers. Male smokers self-reported a trend toward current blue-collar versus white-collar occupation when compared with the nonsmokers. Body weight (77.6 +/- 16.6 kg) did not differ between the smokers and nonsmokers. The male nonsmokers were taller than the male smokers (P < 0.05). All subjects were asymptomatic and had clinically normal spirometry. Compared to male nonsmokers, the male smokers had lower FEF25-75% and FEF75-45% values (P < 0.05). No additional spirometric measurements, including FEV1/FVC, FEV1 and FVC were significantly different. The female smokers did not differ from the female nonsmokers (P < 0.05) in any of the spirometric endpoints measured. Thirteen statistically significant (P < 0.05) correlations involving inflammatory (white blood cells, monocytes, basophils, and PGE1) or immunologic endpoints (IgE) and spirometric measurements were observed in female smokers, female nonsmokers and male nonsmokers. No statistically significant correlations involving immunologic or inflammatory endpoints were observed in the male smokers. A better mechanistic understanding of the observed relationship between elevated hematologic inflammatory endpoints and reduced lung function may provide valuable insight into the clinical significance of these correlations.

A quantitative approach to assessing intercellular communication: studies on cigarette smoke condensates.

Analyses of intercellular communication is useful for assessing the effects of chemical treatment on the function of mammalian cell membranes in vitro. The objective of this study was to quantify and compare the activity of mainstream cigarette smoke condensate (CSC) from tobacco-heating and tobacco-burning cigarettes on both the rate and total amount of intercellular communication in vitro. Lucifer yellow uptake and lactate dehydrogenase release assays were used to evaluate plasma membrane toxicity. Gap junction intercellular communication (GJIC) was determined by quantifying fluorescence redistribution after photobleaching (FRAP) following a 1-hr exposure to concentrations of CSCs which were not toxic to the plasma membrane. GJIC was quantified in rat hepatic epithelial cells (WB cells) and human skin fibroblasts (MSU-2 cells) synchronized in the G1 phase of the cell cycle. In each of the cell types tested, CSC from tobacco-heating cigarettes did not inhibit GJIC at concentrations, where CSC from tobacco-burning cigarettes significantly inhibited both the total amount and the rate of GJIC. These results indicate that mainstream smoke condensate of cigarettes which heat tobacco is less biologically active than mainstream smoke condensate of cigarettes that burn tobacco as determined by in vitro gap junction intercellular communication.

Limitations of the scrape-loading/dye transfer technique to quantify inhibition of gap junctional intercellular communication.

Gap junctional intercellular communication (GJIC) is recognized as playing an important role in normal cell proliferation and development. Chemically induced alteration of GJIC has been proposed to be associated with abnormal cellular growth and/or tumor promotion. Several in vitro assays are currently used to determine the effects of chemicals on GJIC between cultured mammalian cells. One of these assays, the scrape-loading dye transfer (SL/DT) technique, is based on monitoring the transfer of the fluorescent dye Lucifer yellow from one cell into adjacent cells via functional gap junctions. The objective of our study was to evaluate and compare various approaches for quantifying results obtained with the SL/DT technique. Confluent cultures of either WB rat liver epithelial cells or LC-540 rat leydig cells were exposed to the animal tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), solvent (0.1% ethanol), or culture medium for one hour at 37 degrees C prior to analysis of GJIC. Inhibition of dye transfer was clearly evident following TPA exposure. Quantification of this dye transfer was assessed via four approaches: manually counting the number of labeled cells; measuring the distance of dye travel from the scrape line; quantifying the amount of cellular dye uptake; and determining the distribution of dye away from the scrape line. Our results suggest that while the SL/DT technique can be effectively used as a tool to determine the qualitative presence or absence of GJIC, its use in quantifying changes in GJIC following chemical exposure is limited. Since concentration-dependent responses are critical in chemical testing, application of the SL/DT method should be restricted to a screening assay for qualitatively assessing the presence or absence of GJIC.

TGF-beta 1 differentially regulates IL-2 expression and [3H]-thymidine incorporation in CD3 epsilon mAb- and CD28 mAb-activated splenocytes and thymocytes.

Transforming growth factor-beta(1) (TGF-beta(1)) is a critical bifunctional regulator of inflammatory responses. Evidence strongly suggests that these regulatory consequences are, at least in part, a result of profound pleiotropic effects on T lymphocyte effector function. The mechanisms underlying the contradictory biological effects of TGF-beta(1) remain ambiguous. The objective of the present studies was to test the hypothesis that the concentration of TGF-beta(1) and the temporal relationship between activation of the T cell receptor (TCR) and the TGF-beta receptor regulate the effect of TGF-beta(1) on T lymphocyte activation and proliferation. Toward this end, we have quantified the concentration- and time-dependent effect of TGF-beta(1) on interleukin-2 (IL-2) protein secretion as an index of T lymphocyte activation and [3H]-thymidine incorporation as an index of cell proliferation in primary splenocytes and thymocytes. Our results suggest that TGF-beta(1) stimulates IL-2 production at low concentrations (0.1-1 pg/ml) and conversely inhibits IL-2 production at high concentrations (1-10 ng/ml) in CD3epsilon monoclonal antibody (mAb)+/-CD28 mAb-activated splenocytes. Additionally, concentrations of TGF-beta(1) that stimulate IL-2 production in CD3epsilon mAb+CD28 mAb-activated splenocytes concominantly inhibit splenocyte proliferation under similar conditions. Furthermore, we provide evidence suggesting that the effects of TGF-beta(1) on T lymphocytes are dependent upon the temporal relationship between activation of the TCR and the TGF-beta receptor. A time-dependent loss of a stimulatory effect and a concomitant gain of an inhibitory response by TGF-beta(1) on IL-2 production in response to CD3epsilon and CD28 mAbs is observed when TGF-beta(1) is added following T lymphocyte activation. In summary, these data unequivocally demonstrate that the orchestration of paradoxical effects of TGF-beta(1) on T-lymphocyte function is dependent upon the concentration of TGF-beta(1) and the temporal relationship between activation of signaling through the TCR and the TGF-beta receptor. Future mechanistic studies addressing the putative role that these factors play in modulating the effects of TGF-beta(1) on T lymphocyte activity will undoubtedly provide valuable insight towards the pharmacological intervention of inflammatory responses.

Blood parameters associated with atherogenic and thrombogenic risk in smokers and nonsmokers with similar life-styles.

Current evidence indicates that life-style factors can affect the risk of developing cardiovascular disease. The life-style of cigarette smokers, as a group, differs in many ways from that of nonsmokers. Most studies that compare clinical pathologic findings related to atherogenic and thrombogenic risk in smokers and nonsmokers do not adequately control for most of the life-style differences between these two groups. In this study, a number of atherogenic risk factors (cholesterol, low-density lipoprotein, high-density lipoprotein, very low-density lipoprotein, high-density lipoprotein/cholesterol, triglycerides, and glucose) and thrombogenic risk factors (total white blood cell count, total red blood cell count; percent of monocytes, lymphocytes, neutrophils, basophils, and eosinophils; interleukin-1, leukotriene B4, hematocrit, hemoglobin, bilirubin, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, platelet count, prothrombin time, partial thromboplastin time, and fibrinogen) were compared in male and female cigarette smokers and non-smokers who were selected to have approximately similar self-reported life-styles (i.e., food, alcohol, and vitamin consumption and exercise level). However, the smokers (male and female) consumed more coffee (P < 0.05) than the nonsmokers. A trend toward blue-collar versus white collar occupational status was also observed in the male smokers relative to male nonsmokers. Cigarette consumption and urinary cotinine and carboxyhemoglobin levels did not differ between male and female smokers. Atherogenic and thrombogenic values were determined from venous blood samples. No statistically significant (P > 0.05) differences in clinical pathologic findings related to atherogenic risk were observed between the smokers and nonsmokers.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between hydrophobicity of short-chain aliphatic alcohols and their ability to alter plasma membrane integrity.

The quantitative relationship between chemical structure and biological activity has received considerable attention in the fields of pharmacology and drug development. More recently, quantitative structure-activity relationships (QSARs) have been used for predicting chemical toxicity. It has been proposed that alcohols may elicit their toxic effects through hydrophobic interactions with the cellular membrane. The objective of this study was to evaluate the role of hydrophobicity in the loss of membrane integrity following acute exposure to short-chain aliphatic alcohols in rat liver epithelial cells in vitro. The series of alcohols studied included methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 2-butanol, 2-methyl-1-propanol, and 2-methyl-2-propanol. The lactate dehydrogenase (LDH) assay was used to quantify membrane integrity. The logarithm of the octanol/water partition coefficient (log P) was used to quantify hydrophobicity. LDH50 values, representing alcohol concentrations yielding a 50% increase in LDH release relative to untreated controls (i.e., mild disruption of membrane integrity), and EC50 values, representing alcohol concentrations yielding 50% of the maximal release of LDH (i.e., moderate disruption of LDH release), were experimentally determined for each alcohol. The LDH50 and EC50 values were then used to derive the QSAR relationship. The aqueous alcohol concentrations yielding LDH50 or EC50 values ranged from 8.9 x 10(-4) m (LDH50 for octanol) to 3.5 m (EC50 for methanol), and the log P of the alcohols ranged from -0.77 (methanol) to 3.00 (octanol). From these data, we have derived two QSAR equations describing the role of hydrophobicity in the release of LDH from rat liver epithelial cells following a 1-hr alcohol exposure. The QSAR equation for LDH50 values, log (1/LDH50) = 0.896 log P + 0.117 (n = 11, SD = 0.131), was nearly identical to the QSAR equation for EC50 values, log (1/EC50) = 0.893 log P + 0.101 (n = 11, SD = 0.133], suggesting that similar structure-activity relationships exist at both mild and moderate levels of membrane disruption. Our data indicate that an increase in LDH release was positively and linearly correlated with the hydrophobicity (r = 0.993). These data may help predict the potential biological effects of other, as yet untested, aliphatic alcohols and aliphatic alcohol-like compounds (e.g., anesthetics) on the plasma membrane.