Mechanisms involved in A/J mouse lung tumorigenesis induced by inhalation of an environmental tobacco smoke surrogate.

Lung tumors have been reproducibly induced in A/J mice exposed to a surrogate for experimental environmental tobacco smoke (ETSS) in a 5-mo inhalation period followed by 4 mo without further exposure. In order to increase our mechanistic understanding of this model, male mice were whole-body exposed for 6 h/d, 5 d/wk to ETSS with a particulate matter concentration of 100 mg/m(3). Food restriction regimens were included to model or exceed the ETSS-related impairment of body weight development. Half of the mice were pretreated with a single ip injection of urethane to study the effect of the above treatments on lung tumor development induced by this substance. At 5 mo, the tumor response was statistically the same for all groups of non-pretreated mice; however, the expected urethane-induced lung tumorigenesis was significantly inhibited by approximately 25% by ETSS and food restriction. This inhibition was accompanied by a threefold increase in blood corticosterone as a common stress marker for both ETSS and food restriction. At 9 mo, in mice not pretreated, the lung tumor incidence and multiplicity were significantly increased by twofold in the ETSS group; in the urethane-treated groups, the same high tumor multiplicity was reached regardless of previous treatment. The predominant tumor type in all groups was bronchiolo-alveolar adenoma. There was no induction of a specific K-ras mutation pattern by ETSS exposure. These data suggest a stress-induced inhibition of lung tumorigenesis in this model, explaining the need for the posttreatment period.

Toxicological evaluation of an electrically heated cigarette. Part 2: Chemical composition of mainstream smoke.

The chemical composition of mainstream smoke from an electrically heated cigarette (EHC) and that of mainstream smoke from the University of Kentucky Reference Cigarette 1R4F was analyzed. In contrast to the 1R4F, which is a conventional, lit-end cigarette, the EHC is smoked in a microprocessor-controlled lighter with electrical heater elements. The electrical heating causes the tobacco under the heater element to burn at a low temperature during each puff. A comprehensive list of chemical constituents was analyzed in mainstream smoke. The list is a combination of those compounds suggested for analysis in cigarette smoke by a US Consumer Product Safety Commission proposal in 1993, and those cigarette smoke constituents identified by the International Agency on Research on Cancer as being present in cigarette smoke and characterized as carcinogens. The low pyrolysis/combustion temperature of tobacco in the EHC causes distinct shifts in the composition of the smoke compared with a conventional cigarette. A significant drop was seen in the yields of almost all toxicologically relevant constituents. On a per cigarette basis almost two-thirds of the constituents were reduced by at least 80%, whereas on an equal total particulate matter basis about two-thirds of the constituents were reduced by at least 50%, with many constituents reduced by more than 90%.

Evaluation of the potential effects of ingredients added to cigarettes. Part 2: chemical composition of mainstream smoke.

Cigarette mainstream smoke from blended research cigarettes with and without the addition of ingredients was analyzed for its chemical composition. In total, 333 ingredients commonly used in cigarette manufacturing were assigned to three different groups. Each group of ingredients was introduced at a low and a high level to the test cigarettes. The list of the 51 smoke constituents determined is based on those analytes suggested for analysis in a US Consumer Product Safety Commission proposal for low ignition cigarettes and cigarette smoke constituents identified by the International Agency for Research on Cancer as worthy of concern and characterized as carcinogens. An increase in the yield of total particulate matter (TPM) in the range of 13 to 28% relative to the control cigarette without ingredients was observed for all test cigarettes. This was presumably caused by the higher transfer rates of the added ingredients to the smoke compared to the transfer from the tobacco part of the filler. When the yields of individual constituents were normalized to the TPM yields, a reduction in the majority of the constituents was observed when compared to the control. For one of the ingredient groups this reduction was especially high: for phenols a maximum of 70%, for polycyclic aromatic hydrocarbons 50%, and for N-nitrosamines 45%. An increase in the amount relative to TPM was observed for a few smoke constituents: hydrogen cyanide and cadmium (one ingredient group), formaldehyde (one ingredient group), and resorcinol and lead (two ingredient groups). These results are consistent with the lack of any increased activity in the in vitro and in vivo assays in this same series of studies (Food and Chemical Toxicology 2002, 40, 105-111; Food and Chemical Toxicology 2002, 40, 113-131). An overall assessment of our data suggests that these ingredients, when added to the tobacco, do not add to the toxicity of smoke, even at the elevated levels tested in this series of studies.

Hemoglobin adducts in rats chronically exposed to room-aged cigarette sidestream smoke and diesel engine exhaust.

Under controlled conditions with exaggerated concentrations of environmental aerosols, the biologically effective dose markers suggested in the literature as being specific for ETS (i.e., HPB Hb adducts for TSNA exposure) and DEE (i.e., l-aminopyrene Hb adducts for l-nitropyrene exposure) did not respond. A slight but dose-dependent increase in 4-ABP Hb adduct levels was seen in RASS-exposed rats.

Comparison of fresh and room-aged cigarette sidestream smoke in a subchronic inhalation study on rats.

Two experimental types of cigarette sidestream smoke (SS) were compared in a subchronic inhalation study on rats. Fresh SS (FSS) was generated continuously from the reference cigarette 2R1. Room-aged SS (RASS) was generated by aging FSS for 1.5 h in a room with noninert surfaces with materials typically found in residences or offices. Male Sprague-Dawley rats were head-only exposed to three dose levels of each SS type and to filtered, conditioned fresh air (sham-exposure) for 6 h/day, 7 days/week, for 90 days. Room-aging resulted in decreased concentrations of various SS components, e.g., total particulate matter (TPM) and nicotine, while other components, such as carbon monoxide (CO), were not affected. The CO concentrations were 6, 13, and 28 ppm for both SS types. TPM concentrations were between 0.6 and 8.7 micrograms/liter and thus up to 100-fold above the maximum of average concentrations of respiratory suspended particles reported for environmental tobacco smoke. Slight reserve cell hyperplasia in the anterior part of the nose as well as hyperplastic and metaplastic epithelial changes in the larynx were the only observed dose-dependent findings. The metabolism of benzo(a)-pyrene--as a proxy for polycyclic aromatic hydrocarbon metabolism--was induced in the nasal respiratory epithelium and in the lungs while no effect was seen in the nasal olfactory epithelium. The lowest-observed effect level was 6 ppm CO or 0.6 microgram TPM/liter. Most of the effects seen were less expressed in RASS-than in FSS-exposed rats when compared on the basis of the CO concentrations. When compared on the basis of TPM, these effects were equally pronounced for both SS types, suggesting a major role of particulate matter-associated compounds. All findings reverted to sham control levels following a 42-day postinhalation period.

Evidence for peroxynitrite as an oxidative stress-inducing compound of aqueous cigarette smoke fractions.

Previous studies have shown that exposure of Swiss 3T3 cells to mainstream cigarette smoke (CS) trapped in phosphate-buffered saline (smoke-bubbled PBS) resulted in the expression of stress response genes, i.e. haem oxygenase and c-fos, partial inhibition of protein phosphatases 1 and 2A, as well as partial depletion of the cellular glutathione (GSH) pool. Using c-fos gene expression in Swiss 3T3 cells as an indicator for a cellular response against oxidative stress, the following observations are consistent with peroxynitrite as an active principal formed by CS in aqueous solutions: (i) sustained c-fos expression was obtained for smoke-bubbled PBS, peroxynitrite itself and a compound known to stoichiometrically release superoxide and nitric oxide (NO) (3-morpholino-sydnonimine, SIN-1); (ii) c-fos expression in cells exposed to aqueous smoke fractions was inhibited by either the superoxide-scavenging enzyme superoxide dismutase (SOD), in combination with catalase, or the NO-scavenger oxyhaemoglobin (HbO2); and (iii) activation of guanylate cyclase in rat lung cells was observed only when bubbling was performed with filtered smoke and with whole smoke in the presence of SOD/catalase. These results are consistent with a rapid NO-consuming reaction coupled with superoxide-generating properties of the particulate phase of CS. Moreover, (iv) the half-life of the c-fos-inducing activity in smoke-bubbled PBS was found to be <1 h which can be explained by a sustained peroxynitrite formation. Finally, depletion of intracellular thiol levels by smoke-bubbled PBS appears to favour the activation of a redox-sensitive component of the c-fos-inducing pathway.

Mechanism and control of denitrosation of N-nitrosodimethylamine.

The NADPH-dependent microsomal denitrosation of N-nitrosodimethylamine (NDMA) has been investigated using a new procedure which was devised for the determination of nitric oxide under aerobic conditions. On the basis of the results obtained with rat-liver microsomes it is concluded that nitric oxide is formed as a precursor of nitrite in a superoxide dismutase (SOD)-insensitive reaction. The enzyme involved in the denitrosation was found to correspond to the cytochrome P450 isoenzyme responsible for the dealkylation of NDMA. The chemical mechanism of the liberation of nitric oxide is proposed to be of an oxidative nature.

A spectrophotometric assay for superoxide dismutase activities in crude tissue fractions.

A sensitive and reliable assay method was developed to characterize crude cell homogenates and subcellular fractions with regard to their superoxide dismutase (SOD) activities. The determination of SOD activities was based on the well-known spectrophotometric assay introduced by McCord & Fridovich [(1969) J. Biol. Chem. 244, 6049-6055], with partially succinylated (3-carboxypropionylated) rather than native ferricytochrome c as indicating scavenger. Partial succinylation of cytochrome c resulted in minimization of interference associated with the interaction of cytochrome c with mitochondrial cytochrome c oxidase or cytochrome c reductases. The further increase in specificity, with regard to exclusion of cytochrome c oxidase interference, gained as a consequence of the high pH of 10 enabled the analysis of samples as rich in cytochrome c oxidase activity as the mitochondrial fraction in the presence or absence of membrane-disrupting detergents. Linear relationships for the dependence of the SOD activities with protein concentration were obtained with rat liver homogenate, mitochondrial and microsomal fractions, indicating negligible interference. Furthermore, by choosing a high pH for the assay medium, a 4-fold increase in sensitivity compared with the classical SOD assay, carried out at pH 7.8, was gained as well as a more precise resolution of Cu/Zn-SOD and Mn-SOD by 2 mM-KCN in samples with a high ratio of Mn-SOD to Cu/Zn-SOD, such as mitochondria. The complete trapping of the O2.- radicals, which was more feasible at pH 10 than at pH 7.8, enabled the application of a simple equation derived for the calculation of appropriately defined units of SOD activity from a single experiment.

Metabolic activation of carbon tetrachloride: induction of cytochrome P-450 with phenobarbital or 3-methylcholanthrene and its effect on covalent binding.

Anaerobic incubation of [14C]carbon tetrachloride with normal rat liver microsomes and microsomes from rats treated with the inducers phenobarbital (PB) and 3-methylcholanthrene (MC) reveals distinct differences in metabolic activation. While the increase in CO-binding pigment is comparable for both inducers, metabolism of CC14 is enhanced only by PB-induction; MC-induced microsomes are equivalent to microsomes from untreated animals. Sodium dodecyl sulphate (SDS)-electrophoresis of microsomal proteins confirms the expected increase at 52 000 daltons (cytochrome P-450 PB) on PB-induction, at 56 000 daltons (cytochrome P-450MC) on MC-induction; after anaerobic incubation with [14C]CC14 the electrophoretic pattern is largely unchange. The highly reactive radical intermediates of CC14-metabolism should attack the closest possible partner. Most of protein-bound radioactivity is located in the mass range between 47 000 and 54 000 daltons, indicating that cytochrome P-450PB is the isoenzyme mainly responsible for CC14-activation; cytochrome P-450MC plays virtually no role in metabolic activation. The direct participation of NADPH-cytochrome P-450 reductase appears unlikely, since the specific binding to proteins in the corresponding mass range is not elevated. A significant percentage of label is attached to proteins at 120 000 daltons and above, presumably oligomers of cytochrome P-450 apoprotein.