Genetic toxicology studies comparing the activity of sidestream smoke from cigarettes which burn or only heat tobacco

The results of in vitro genetic toxicology studies of sidestream cigarette smoke (SSCS) from cigarettes which heat but do not burn tobacco were compared to those of sidestream smoke from cigarettes which burn tobacco. SSCSs from 5 cigarettes were compared. Three of the cigarettes, the Kentucky reference research cigarette (1R4F), a commercially available ultra-low-tar brand (ULT) and a commercially available ultra-low-tar menthol brand (ULT-menthol) burn tobacco while two of the cigarettes, a regular (TEST) and a menthol (TEST-menthol) heat tobacco. SSCSs from all cigarettes were prepared by identical techniques, which involved collecting sidestream smoke particulate matter on Cambridge filter pads and combining the particulate matter with the vapor-phase materials collected by bubbling the smoke exiting the Cambridge pad through DMSO. The SSCSs obtained (equivalent to 0.4 cigarettes/ml DMSO) were evaluated at identical concentrations in an in vitro genetic toxicology test battery. SSCS from 1R4F, ULT and ULT-menthol cigarettes produced positive results in Ames bacterial strains TA98, TA100, TA1537 and TA1538 in the presence of metabolic activation (S9 from Aroclor-induced rat liver) but negative results in strain TA1535. In the absence of metabolic activation, 1R4F, ULT and ULT-menthol SSCSs were not significantly mutagenic. TEST and TEST-menthol SSCSs produced negative results in all 5 bacterial strains, both with and without metabolic activation. SSCS from 1R4F, ULT and ULT-menthol cigarettes produced positive results in the CHO chromosomal aberration assay and in the CHO sister-chromatid exchange assay both with and without metabolic activation while TEST and TEST-menthol SSCSs produced negative results in both assays, either with or without metabolic activation. The SSCSs from 1R4F, ULT and ULT-menthol cigarettes were weakly positive in inducing DNA repair in cultured rat hepatocytes while TEST and TEST-menthol SSCSs were negative in this assay. All 5 SSCSs were nonmutagenic in the CHO-HGPRT assay both with and without metabolic activation. SSCSs from the 1R4F, ULT and ULT-menthol cigarettes were cytotoxic in the CHO-HGPRT assay, both with and without metabolic activation, while TEST and TEST-menthol SSCSs were not cytotoxic under either condition. These results demonstrate that sidestream smoke from cigarettes which heat but do not burn tobacco (TEST and TEST-menthol) was neither genotoxic nor cytotoxic under conditions where sidestream smoke from cigarettes which burn tobacco (1R4F, ULT and ULT-menthol) was genotoxic and/or cytotoxic in a concentration-dependent manner.     

A comparative study by electron paramagnetic resonance of free radical species in the mainstream and sidestream smoke of cigarettes with conventional acetate filters and 'bio-filters'

Tobacco smoking is the most important extrinsic cause, after the diet, for increasing morbidity and mortality in humans. Unless current tobacco smoking patterns in industrialised and non-industrialised countries change, cigarettes will kill prematurely 10 million people a year by 2025. Greece is at the top of the list of European countries in cigarette consumption. In 1997, a Greek tobacco company introduced a new 'bio-filter' (BF) claiming that it reduces substantially the risks of smoking. In a recent publication [Deliconstantinos G, Villiotou V, Stavrides J. Scavenging effects of hemoglobin and related heme containing compounds on nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke. A new method for protection against the dangerous cigarette constituents. Anticancer Res 1994; 14: 2717-2726] it was claimed that the new 'bio-filter' (activated carbon impregnated with dry hemoglobin) reduces certain toxic substances and oxidants (like NO, CO, NOx, H2O2, aldehydes, trace elements and nitroso-compounds) in the gas-phase of the mainstream smoke. We have investigated by electron paramagnetic resonance (EPR) the mainstream and sidestream smoke of the BF cigarette, in comparison with three other cigarettes with similar tar and nicotine contents, that have conventional acetate filters. We found that BF cigarette smoke has similar tar radical species with the same intensity EPR signals to those of the other cigarettes. The ability of the aqueous cigarette tar extracts to produce hydroxyl radicals (HO*), which were spin trapped by DMPO, was very similar to, or even higher than, the other 3 brands. The gas-phase of the mainstream smoke of the BF cigarette showed a 30-35% reduction in the production of oxygen-centered radicals (spin trapped with PBN). In the case of the sidestream smoke, BF cigarettes produced substantially higher concentrations of gas-phase radicals, compared to the other brands. These results suggest that BF is partially effective at removing some of the gas-phase oxidants but not effective in the reduction of tar and its radical species in the mainstream and sidestream smoke. It is well known from epidemiological studies that tar content is strongly associated with increasing risk to smokers of lung cancer. In our experiments, BF cigarettes produce a higher amount of tar and stable free radical species than the other 3 brands in the sidestream smoke (between puffs), thus potentially increasing risk to the smoker and passive smoker.     

Aqueous cigarette tar extracts damage human alpha-1-proteinase inhibitor

The elastase inhibitory capacity (EIC) of human alpha-1-proteinase inhibitor (alpha 1PI) is severely compromised by aqueous cigarette tar extract (ACTE). An aqueous extract of the tar from two cigarettes causes a loss of EIC of at least 60% in 24 h at 37 degrees C (pH 7.4) and the damaging capability of the ACTE is retained for many hours. Hydrogen peroxide appears to be an essential component of the mechanism by which ACTE damages alpha 1 PI, since catalase substantially protects alpha 1PI from ACTE-mediated damage. Only mild protection is offered by 10 mM diethylenetriamine pentaacetic acid, indicating only a minor role for transition metal ions in the alpha 1PI-damaging process. Hydroxyl radicals are unlikely agents of alpha 1PI damage in the ACTE system, as judged from hydroxyl radical scavenger studies. Ascorbate and various thiols offer protection to different degrees, dependent on the incubation conditions. Of several amino acids tested, cysteine and methionine (but not methionine sulfoxide) are the only two that protect alpha 1PI. We suggest that components of cigarette smoke particulate matter extracted into the aqueous lung fluid environment may cause local deficiencies in alpha 1PI in smokers' lungs.     

Mutagenicity of native cigarette mainstream smoke and its gas/vapour phase by use of different tester strains and cigarettes in a modified Ames assay

The "Bacterial Reverse Mutation Assay" is generally accepted to analyse the genotoxic capacity of single compounds or complex mixtures such as cigarette-smoke condensates. With an adapted and modified Ames assay, the mutagenicity of native cigarette mainstream whole smoke (WS) and its gas/vapour phase (GVP) was studied. The bacteria were directly exposed to the smoke in a CULTEX1 system closely connected to a smoking robot (VC10). A variety of standard tester strains (TA98, TA100, TA1535, TA1537, TA1538, TA102, WP2uvrApKM101) and descendants of TA98 (YG1021, YG1024, YG1041) and TA100 (YG1026, YG1029 and YG1042) were exposed to whole and filtered smoke of the research cigarette K2R4F to find the most sensitive strains for analysing the mutagenic activity of these test atmospheres. Mutagenicity of WS was detected by TA98, TA100 and their YG descendant strains as well as by WP2uvrApKM101 in the presence of S9 mix. The GVP induced a mutagenic signal in TA100, YG1029 and YG1042 and WP2uvrApKM101 only in the absence of S9 mix. To detect mutagenicity in WS the presence of the plasmid pKM101 is required and a frame-shift mutation is more effective than a missense mutation. To detect mutagenicity in GVP, the presence of the plasmid pKM101 and a missense mutation are required. The differentiating capacity of this modified Ames assay was demonstrated by exposing strain TA98 to WS and TA100 to the GVP of cigarettes with different tar content. The mutagenic activity of WS and the GVP increased with rising tar content of the cigarettes with two exceptions in WS. Thus, the concept of tar content alone is misleading and does not reflect the mutagenic activity of a cigarette.     

Biological effects of cigarette smoke, wood smoke, and the smoke from plastics: The use of electron spin resonance

This review compares and contrasts the chemistry of cigarette smoke, wood smoke, and the smoke from plastics and building materials that is inhaled by persons trapped in fires. Cigarette smoke produces cancer, emphysema, and other diseases after a delay of years. Acute exposure to smoke in a fire can produce a loss of lung function and death after a delay of days or weeks. Tobacco smoke and the smoke inhaled in a burning building have some similarities from a chemical viewpoint. For example, both contain high concentrations of CO and other combustion products. In addition, both contain high concentrations of free radicals, and our laboratory has studied these free radicals, largely by electron spin resonance (ESR) methods, for about 15 years. This article reviews what is known about the radicals present in these different types of smokes and soots and tars and summarizes the evidence that suggests these radicals could be involved in cigarette-induced pathology and smoke-inhalation deaths. The combustion of all organic materials produces radicals, but (with the exception of the smoke from perfluoropolymers) the radicals that are detected by ESR methods (and thus the radicals that would reach the lungs) are not those that arise in the combustion process. Rather they arise from chemical reactions that occur in the smoke itself. Thus, a knowledge of the chemistry of the smoke is necessary to understand the nature of the radicals formed. Even materials as similar as cigarettes and wood (cellulose) produce smoke that contains radicals with very different lifetimes and chemical characteristics, and mechanistic rationales for this are discussed. Cigarette tar contains a semiquinone radical that is infinitely stable and can be directly observed by ESR. Aqueous extracts of cigarette tar, which contain this radical, reduce oxygen to superoxide and thus produce both hydrogen peroxide and the hydroxyl radical. These solutions both oxidize alpha-1-proteinase inhibitor (a1PI) and nick DNA. Because of the potential role of radicals in smoke-inhalation injury, we suggest that antioxidant therapy (such as use of an inhaler for persons brought out of a burning building) might prove efficacious.     

Inhibition of lactate dehydrogenase in cultured SIRC cells by cigarette smoke

Summary SIRC cell monolayer cultures were exposed to whole smoke from a mid tar and nicotine level research cigarette (ASFC, 72 puffs), or from a high tar and nicotine level reference cigarette (Kentucky 2R1, 48 puffs) over a period of 65 days. The activity and distribution of lactate dehydrogenase (LDH) in the cells were investigated, and the electrophoretic characteristics of its isozymes studied. Cell morphology was examined by light microscopy and by transmission- and scanning electron microscopy.LDH activity was reduced by exposure to smoke from both cigarette types, the greater inhibitory effect being produced by that of the Kentucky cigarette. In addition, cells exposed to this high tar and nicotine smoke displayed intramitochondrial granules which were larger and more numerous than those found in cells exposed to the mid tar and nicotine smoke, or in the control cells. It is speculated that cation accumulation in the mitochondria may be involved in the observed inhibition of LDH activity.Supported by a research grant from the ASFC (Association Suisse des Fabricants de Cigarettes), Switzerland     

The role of glutathione in the toxicity of smoke condensates from cigarettes that burn or heat tobacco

Inhalation of cigarette smoke aerosol via active smoking is associated with the development of pulmonary inflammation. The cytotoxic potential of cigarette smoke has been hypothetically related to development of pulmonary inflammation since the release of intracellular contents from dead and dying cells has been reported to induce inflammatory foci. In this study, cigarette smoke condensates (CSCs) were prepared from Kentucky 1R4F reference cigarettes and cigarettes that primarily heat tobacco (Eclipse). The two CSCs were then compared for their ability to induce killing in human-hamster A_L hybrid cells. CSCs prepared from Eclipse were much less cytotoxic than those prepared from reference cigarettes. At 60 μg CSC/ml culture medium, survival for CSC from Eclipse cigarettes was approximately 70% compared with 1% for CSC from burned K1R4F cigarettes. The observed reduction in CSC-Eclipse cytotoxicity toward these mammalian cells is consistent with the previously published observation of a 30% decline in pulmonary white cell count and 40% reduction in visual bronchitis index in human smokers who switched to Eclipse for 2 months. Results with N-acetylcysteine and buthionine-S-R-sulfoximine indicate that glutathione markedly reduces the cytoxicity of both CSCs.     

DNA damage induced by cigarette smoke condensate in vitro as assayed by 32P-postlabeling. Comparison with cigarette smoke-associated DNA adduct profiles in vivo.

Cigarette smoke induces a multitude of bulky/aromatic DNA adducts in vivo as revealed by 32P-postlabeling assay. The formation of such adducts is thought to involve metabolic activation of aromatic chemicals especially polycyclic aromatic hydrocarbons (PAHs) present in tumor-initiating cigarette tar fractions, via cytochrome P450-associated monooxygenases. Because radicals are present in both the gas and particulate (tar) phase of cigarette smoke and in aqueous extracts of cigarette smoke condensate (CSC), we addressed the question as to whether cytochrome P450-independent, possibly free radical-mediated reactions may contribute, also, to formation of cigarette smoke-associated bulky DNA adducts. Rat-lung DNA was incubated with aqueous extracts of CSC in the absence of microsomes under various conditions and analyzed by 32P-postlabeling. Radioactively labeled bulky reaction products were found to accumulate in a time- and CSC concentration-dependent manner. The resulting chromatographic profiles resembled cigarette smoke-associated DNA-adduct patterns observed in vivo. Pretreatment of aqueous CSC extract with radical scavengers/reducing agents (ascorbic acid, glutathione) diminished adduct formation in a concentration-dependent manner. Adduct formation in vitro may involve oxygen-free radicals, which are known to be present in aqueous CSC extracts and could (i) attack DNA directly to produce bulky adducts, (ii) induce radical sites on DNA covalently binding CSC components, or (iii) convert CSC components to DNA-reactive electrophiles. In addition, DNA may react with direct-acting mutagens in CSC. Adduct fractions derived from in vitro and in vivo experiments showed similar chromatographic behavior, suggesting that metabolic activation as well as processes not involving metabolism lead to formation of smoking-induced bulky DNA adducts in vivo.     

A protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking (5,6,7,12). Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens(9,10). Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung(3). Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke(4). A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke(14). However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine(8). In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy(1,2,14). We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively(11,13).     

Cigarette smoke-induced pulmonary inflammation is TLR4/MyD88 and IL-1R1/MyD88 signaling dependent

Acute cigarette smoke exposure of the airways (two cigarettes twice daily for three days) induces acute inflammation in mice. In this study, we show that airway inflammation is dependent on Toll-like receptor 4 and IL-1R1 signaling. Cigarette smoke induced a significant recruitment of neutrophils in the bronchoalveolar space and pulmonary parenchyma, which was reduced in TLR4-, MyD88-, and IL-1R1-deficient mice. Diminished neutrophil influx was associated with reduced IL-1, IL-6, and keratinocyte-derived chemokine levels and matrix metalloproteinase-9 activity in the bronchoalveolar space. Further, cigarette smoke condensate (CSC) induced a macrophage proinflammatory response in vitro, which was dependent on MyD88, IL-1R1, and TLR4 signaling, but not attributable to LPS. Heat shock protein 70, a known TLR4 agonist, was induced in the airways upon smoke exposure, which probably activates the innate immune system via TLR4/MyD88, resulting in airway inflammation. CSC-activated macrophages released mature IL-1beta only in presence of ATP, whereas CSC alone promoted the TLR4/MyD88 signaling dependent production of IL-1alpha and pro-IL-1beta implicating cooperation between TLRs and the inflammasome. In conclusion, acute cigarette exposure results in LPS-independent TLR4 activation, leading to IL-1 production and IL-1R1 signaling, which is crucial for cigarette smoke induced inflammation leading to chronic obstructive pulmonary disease with emphysema.     

Mechanisms of cigarette smoke toxicity: the inactivation of human alpha-1-proteinase inhibitor by nitric oxide/isoprene mixtures in air

A mixture of nitric oxide (NO) and isoprene in air has been studied as a model for gas-phase cigarette smoke. We have shown that this model system duplicates many of the properties of cigarette smoke including the inactivation of human alpha-1-proteinase inhibitor (a1PI). In this study, buffered solutions of a1PI were exposed to puffs of air containing 300 ppm NO and 400 ppm isoprene. Bubbling of the NO/air/isoprene gas stream directly through buffered protein solutions causes a1PI to undergo a fast loss of inhibitory capacity. This fast inactivation is not observed when a1PI is exposed to aqueous extracts of the NO/air/isoprene mixture. Both direct exposure and exposure to aqueous extracts, however, cause a1PI to undergo a slow loss of activity that continues for several days as the protein is incubated in the buffer solutions. Gas-phase cigarette smoke has already been shown to cause this same two-phase inactivation of a1PI. The inactivation of a1PI by the model system is dependent on the presence of oxygen in the gas stream, suggesting that the oxidation of nitric oxide to nitrogen dioxide in air is involved in the formation of the inactivating species. The nature of these species remains to be determined; however, small alkoxyl or peroxyl radicals (such as are spin-trappable from gas-phase smoke as well as from the NO/air/isoprene system) do not appear to inactivate a1PI. One possibility is that the inactivating species are metastable compounds formed by radical processes in the gas phase of both cigarette smoke and our model system. Our data suggest that one possible class of species is peroxynitrates.     

Low-tar medium-nicotine cigarettes: a new approach to safer smoking.

The logic of expecting people who cannot stop smoking to switch to cigarettes that have hardly any nicotine is questionable. Tar and nicotine yields of cigarettes available in Britain today correlate 0-93, and further reduction of tar intake is limited by the reluctance of smokers to tolerate similar reductions in nicotine. A new approach would be to aim at lowering tar yields of cigarettes from the present average of 18 mg to around 6 mg but maintaining nicotine yields at around 1-0 to 1-2 mg, which would be acceptable to most smokers. This approach requires that emphasis be placed on tar: nicotine ratios as well as on the absolute yields. These ratios for brands on sale in Britain today average 14-2 and range from 9-6 to 20-8. They provide an additional guide for comparing the relative harmfulness of different brands. For example, 35% of cigarette smokers in Britain smoke either Embassy Filter or Players No 6 Filter; by changing to John Player Carlton King Size they could reduce their tar intake by more than 20% without having to suffer any nicotine deprivation.     

A Simple and Rapid Method for Standard Preparation of Gas Phase Extract of Cigarette Smoke

Cigarette smoke consists of tar and gas phase: the latter is toxicologically important because it can pass through lung alveolar epithelium to enter the circulation. Here we attempt to establish a standard method for preparation of gas phase extract of cigarette smoke (CSE). CSE was prepared by continuously sucking cigarette smoke through a Cambridge filter to remove tar, followed by bubbling it into phosphate-buffered saline (PBS). An increase in dry weight of the filter was defined as tar weight. Characteristically, concentrations of CSEs were represented as virtual tar concentrations, assuming that tar on the filter was dissolved in PBS. CSEs prepared from smaller numbers of cigarettes (original tar concentrations ≤15 mg/ml) showed similar concentration-response curves for cytotoxicity versus virtual tar concentrations, but with CSEs from larger numbers (tar ≥20 mg/ml), the curves were shifted rightward. Accordingly, the cytotoxic activity was detected in PBS of the second reservoir downstream of the first one with larger numbers of cigarettes. CSEs prepared from various cigarette brands showed comparable concentration-response curves for cytotoxicity. Two types of CSEs prepared by continuous and puff smoking protocols were similar regarding concentration-response curves for cytotoxicity, pharmacology of their cytotoxicity, and concentrations of cytotoxic compounds. These data show that concentrations of CSEs expressed by virtual tar concentrations can be a reference value to normalize their cytotoxicity, irrespective of numbers of combusted cigarettes, cigarette brands and smoking protocols, if original tar concentrations are ≤15 mg/ml.     

Early specific free radical-related cytotoxicity of gas phase cigarette smoke and its paradoxical temporary inhibition by tar: An electron paramagnetic resonance study with the spin trap DEPMPO

Electron paramagnetic resonance (EPR) spin trapping studies demonstrated aqueous tar particulate matter (TPM) and gas phase cigarette smoke (GPCS) to behave as different sources of free radicals in cigarette smoke (CS) but their cytotoxic implications have been only assessed in CS due to its relevance to the natural smoking process. Using a sensitive spin trapping detection with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), this study compared the respective roles of CS- and GPCS-derived free radicals on smoke-induced cytotoxicity and lipid peroxidation of filtered and unfiltered, machine-smoked experimental and reference cigarettes yielding a wide range of TPM yields. In buffer bubbled with CS the DEPMPO/superoxide spin adduct was the major detected nitroxide. Use of appropriate control experiments with nitric oxide radical (NO*) or carbonyl sulfide, and a computer analysis of spin adduct diastereoisomery showed that the hydroxyl radical (HO*) adduct of DEPMPO seen in GPCS-bubbled was rather related to metal-catalyzed nucleophilic synthesis than to direct HO* trapping. Unexpectedly a protective effect of TPM on murine 3T3 fibroblasts was observed in early (<3h) free radical-, GPCS-induced cell death, and carbon filtering decreased free radical formation, toxicity and lipid peroxidation in three cell lines (including human epithelial lung cells) challenged with GPCS. These results highlight an acute, free radical-dependent, harmful mechanism specific to the GPCS phase, possibly involving NO* chemistry, whose physical or chemical control may be of great interest with the aim of reducing the toxicity of smoke.     

Biomarkers of exposure and potential harm in adult smokers of 3-7 mg tar yield (Federal Trade Commission) cigarettes and in adult non-smokers.

The paper reports levels of 24-h urine nicotine and five of its major metabolites (expressed as nicotine-equivalents) and blood carboxyhaemoglobin as biomarkers of exposure to particulate- and gas-phase cigarette smoke, respectively, from an exploratory pilot study of adult smokers of 3.0-6.9 mg tar delivery (Federal Trade Commission (FTC) method) cigarettes. On multiple occasions over 6 weeks, blood high-sensitivity C-reactive protein (hs-CRP), fibrinogen, HDL- and LDL-cholesterol, and 24-h urine 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha) and 11-dehydro-thromboxane B2 (11-dehydro-TxB2) were also evaluated as biomarkers of potential harm. All the biomarkers examined, except for LDL-cholesterol, discriminated with high sensitivity and specificity between adult smokers and non-smokers overall. Except for HDL-cholesterol, all biomarker medians were greater in adult smokers than in non-smokers: urine nicotine-equivalents 64.514 versus < 0.034 nmol mg-1 creatinine (p<0.001), carboxyhaemoglobin 4.0 versus 0.4% saturation (p<0.001), hs-CRP 0.27 versus 0.12 mg dl-1 (p=0.05), fibrinogen 292 versus 248 mg dl-1 (p<0.001), HDL-cholesterol 46 versus 53 mg dl-1 (p=0.003), LDL-cholesterol 119 versus 109 mg dl-1 (p=0.18), urine 8-epi-PGF2alpha 1935 versus 1034 pg mg-1 creatinine (p<0.001) and urine 11-dehydro-TxB2 973 versus 710 pg mg-1 creatinine (p<0.001). All the biomarkers of exposure and most of the biomarkers of potential harm showed no time of sampling (by visit week) effect.     

Relation of nicotine yield of cigarettes to blood nicotine concentrations in smokers.

Blood nicotine and carboxyhaemoglobin (COHb) concentrations were studied in 330 smokers (206 women and 124 men). Blood nicotine concentrations in individual smokers varied from 25 to 444 nmol/l (4 to 72 ng/ml). The average concentration, 203 nmol/l (33 ng/ml), was the same in the men and the women, although cigarette consumption was higher in the men. Despite large differences in nicotine yield, there was no relation between blood nicotine concentration and the type of cigarette smoked: smokers of plain, untipped cigarettes (1.9 mg nicotine), cigarettes with unventilated filters (1.3 mg nicotine), and cigarettes with ventilated filters (0.8 mg nicotine) had similar blood nicotine concentrations. Cigarette consumption was also similar in these three groups. The correlation between blood nicotine concentration and nicotine yield of cigarette, though significant, was low (0.21, p < 0.001), showing that the nicotine yield of the cigarettes accounted for only 4.4% of the variation in blood nicotine concentrations. Similarly, the low correlation of 0.30 between COHb concentration and cigarette consumption suggests that cigarette consumption accounted for only 9% of the variation in the amount of smoke taken into the smokers'' lungs. These results suggest that the assumed health advantage of switching to lower-tar and lower-nicotine cigarettes may be largely offset by the tendency of smokers to compensate by increasing inhalation. The findings of epidemiological studies showing lower risks with filter-tipped cigarettes may be attributable to other factors such as biases in the samples and changes in the quality and carcinogenicity of tobacco tar, rather than to reduced tar intake.     

The inhibitory effect of extracts of cigarette tar on electron transport of mitochondria and submitochondrial particles.

Acetonitrile extracts of cigarette tar inhibit state 3 and state 4 respiration of intact mitochondria. Exposure of respiring submitochondrial particles to acetonitrile extracts of cigarette tar results in a dose-dependent inhibition of oxygen consumption and reduced nicotinamide adenine dinucleotide (NADH) oxidation. This inhibition was not due to a solvent effect since acetonitrile alone did not alter oxygen consumption or NADH oxidation. Intact mitochondria are less sensitive to extracts of tar than submitochondrial particles. The NADH-ubiquinone (Q) reductase complex is more sensitive to inhibition by tar extract than the succinate-Q reductase and cytochrome complexes. Nicotine or catechol did not inhibit respiration of intact mitochondria. Treatment of submitochondrial particles with cigarette tar results in the formation of hydroxyl radicals, detected by electron spin resonance (ESR) spin trapping. The ESR signal attributable to the hydroxyl radical spin adduct requires the presence of NADH and is completely abolished by catalase and to a lesser extent superoxide dismutase (SOD). Catalase and SOD did not protect the mitochondrial respiratory chain from inhibition by tar extract, indicating that the radicals detected by ESR spin trapping are not responsible for the inhibition of the electron transport. We propose that tar causes at least two effects: (1) Tar components interact with the electron transport chain and inhibit electron flow, and (2) tar components interact with the electron transport chain, ultimately to form hydroxyl radicals.     

Prolonged exposure to cigarette smoke blocks the neurotoxicity induced by kainic acid in rats

We examined the effects of cigarette smoke (CS) on three parameters associated with kainic acid (KA)-induced neurotoxicity: seizure activity, cell loss in the hippocampus, and increased Fos-related antigen (FRA) expression. Animals were exposed to the main stream of CS from 15 Kentucky 2R1F research cigarettes containing 28.6 mg tar and 1.74 mg nicotine per cigarette, for 10 min a day, 6 days per week, for 4 weeks, using an automatic smoking machine. KA administration (10 mg/kg, i.p.) produced robust behavioral convulsions lasting 4-5 h. Pre-exposure to CS significantly reduced the seizures, mortality, and severe loss of cells in regions CA1 and CA3 of the hippocampus after KA administration. Consistently, pre-exposure to CS significantly attenuated the KA-induced increased FRA immunoreactivity in the hippocampus. In contrast, pretreatment with central nicotinic antagonist, mecamylamine (2 or 10 mg/kg, i.p.) blocked the neuroprotective effects mediated by CS in a dose-dependent manner. These results indicate that CS exposure provides neuroprotection against the KA insult via nicotinic receptor activation.     

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.     

Enhancement by cigarette smoke extract of the radical formation in a reaction mixture of 13-hydroperoxide octadecadienoic acid and ferric ions

The effects of cigarette smoke extract on radical formation were examined in reaction mixtures containing 13-hydroperoxide octadecadienoic acid (13-HPODE), FeCl3, cigarette smoke extract, ethylenediaminetetraacetic acid (EDTA), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN), and phosphate buffer (pH 7.4). Cigarette smoke extract enhanced the formation of both 7-carboxyheptyl and pentyl radicals in the reaction. Ferric ions were reduced in the reaction mixture, suggesting that cigarette smoke extract enhances the formation of 7-carboxyheptyl and pentyl radicals by reducing ferric irons. Although there is a large body of evidence supporting the involvement of radicals such as the semiquinone radical, hydroxyl radical, superoxide radical, nitric oxide radicals in smoking-related diseases, the enhancement by cigarette smoke of lipid-derived radical formation, which we first report here, may be one of the other causes of smoking-related diseases.