Toxicological assessment of kretek cigarettes. Part 7: the impact of ingredients added to kretek cigarettes on inhalation toxicity.

The biological activity of mainstream smoke from experimental kretek cigarettes with and without three mixes of ingredients was assessed in a 90-day rat inhalation study and in a 4-day in vivo micronucleus assay. 350 ingredients, commonly used in various combinations and in a limited number in a given brand in the manufacture of marketed kretek cigarettes, were applied at a low and a high target level to test cigarettes with a typical Indonesian blend of tobaccos and cloves. In the 90-day inhalation study, effects commonly seen in rat inhalation studies with mainstream smoke were observed. In general, no ingredients-related histopathological changes were found in the respiratory tract. In the 4-day micronucleus assay exposure of male rats to mainstream smoke from the test cigarettes containing any of the three mixes did not increase the proportions of micronucleated cells in peripheral blood and bone marrow over the proportion of micronucleated cells in the control group. Based on the results of these studies, it can be concluded that the addition of ingredients commonly used in the manufacture of kretek cigarettes did not change the overall in vivo toxicity profile of the mainstream smoke.

Toxicological assessment of kretek cigarettes Part 3: kretek and American-blended cigarettes, inhalation toxicity.

A typical Indonesian kretek cigarette brand and an experimental kretek reference cigarette were compared to the reference cigarette 2R4F in two 90-day inhalation studies. Male and female rats were exposed nose-only to mainstream smoke for 6 hours daily, for 90 consecutive days. Biological endpoints were assessed according to OECD guideline 413, with special emphasis on respiratory tract histopathology and on lung inflammation (broncho-alveolar lavage fluid levels of neutrophils, macrophages and lymphocytes). Histopathological alterations included: in the nose, hyperplasia and squamous metaplasia of the respiratory epithelium and squamous metaplasia and atrophy of the olfactory epithelium; in the larynx, epithelial squamous metaplasia and hyperplasia; in the lungs, accumulation of macrophages in alveoli and goblet cell hyperplasia in bronchial epithelium. The findings were qualitatively consistent with observations from previous similar studies on conventional cigarettes. Compared to 2R4F cigarette, however, kretek smoke exposure was associated with a pronounced attenuation of pulmonary inflammation and less severe histopathological changes in the respiratory tract. Neutrophilic inflammation was also significantly lower (>70%). These results are consistent with the observations made on smoke chemistry and in vitro toxicology. They do not support any increased toxicity of the smoke of kretek cigarettes compared to conventional American-blended cigarettes.

Toxicological assessment of kretek cigarettes Part 5: mechanistic investigations, inhalation toxicity.

The biological effects of mainstream smoke (MS) from Indonesian-blended cigarettes with and without added cloves, cloves extracted with hot ethanol, and extracted cloves replenished with eugenol or clove oil were assessed in a 90-day inhalation study in rats. A separate 35-day inhalation study in rats was performed with MS from American-blended cigarettes with 0%, 2.5%, 5% or 10% added eugenol. Effects commonly seen in inhalation studies with MS were observed. These included histopathological changes indicative of irritation in the entire respiratory tract and inflammatory responses in the lung. Adding cloves to American- or Indonesian-blended cigarettes reduced the inflammatory response in the lung but with no difference between the two blend types. When the clove oil was extracted (∼ 75% reduction of eugenol achieved) from cloves, the inflammatory response in the lung was still reduced similarly to whole cloves but the severity of histopathological changes in the upper respiratory tract was less reduced. Add back of clove oil or pure eugenol reduced this response to a level similar to what was seen with whole cloves. When eugenol was added to American-blended cigarettes, similar findings of reduced lung inflammation and severity of histopathological changes in respiratory the tract was confirmed. These studies demonstrate a clear effect of cloves, and in particular eugenol, in explaining these findings.

Reduced toxicological activity of cigarette smoke by the addition of ammonia magnesium phosphate to the paper of an electrically heated cigarette: subchronic inhalation toxicology.

Cigarette smoke is a complex chemical mixture that causes a variety of diseases, such as lung cancer. With the electrically heated cigarette smoking system (EHCSS), temperatures are applied to the tobacco below those found in conventional cigarettes, resulting in less combustion, reduced yields of some smoke constituents, and decreased activity in some standard toxicological tests. The first generation of electrically heated cigarettes (EHC) also resulted in increased formaldehyde yields; therefore, a second generation of EHC was developed with ammonium magnesium phosphate (AMP) in the cigarette paper in part to address this increase. The toxicological activity of mainstream smoke from these two generations of EHC and of a conventional reference cigarette was investigated in two studies in rats: a standard 90-day inhalation toxicity study and a 35-day inhalation study focusing on lung inflammation. Many of the typical smoke exposure-related changes were found to be less pronounced after exposure to smoke from the second-generation EHC with AMP than to smoke from the first-generation EHC or the conventional reference cigarette, when compared on a particulate matter or nicotine basis. Differences between the EHC without AMP and the conventional reference cigarette were not as prominent. Overall, AMP incorporated in the EHC cigarette paper reduced the inhalation toxicity of the EHCSS more than expected based on the observed reduction in aldehyde yields.

Toxicological evaluation of an electrically heated cigarette. Part 4: Subchronic inhalation toxicology.

The biological activity of mainstream smoke from an electrically heated cigarette (EHC) with controlled combustion and from the University of Kentucky Reference Cigarette 1R4F was determined in Sprague Dawley rats exposed nose-only for 90 days, 6 h a day, 7 days per week. For an equivalent response comparison between the two cigarette types, two doses were chosen for the EHC where the anticipated results were in the dynamic range of the 1R4F dose-response curve (four concentrations) for most end points. The number of cigarettes smoked per m(3) of diluted smoke resulted in total particulate matter concentrations of 40 and 90 microg l (-1) for the EHC and 40-170 microg l (-1) for the 1R4F. Biomonitoring indicated achievement of target doses. Mainstream smoke yields were lower for the EHC, with the exception of formaldehyde. No smoke-related mortality, remarkable in-life observations or abnormal gross pathological findings were observed. Smoke- and dose-related clinical pathology and organ weight changes included: increases in segmented neutrophils, some liver parameters and lung and adrenal weight relative to body weight; and decreases in lymphocytes, glucose concentration and spleen weight. Smoke-related histopathological findings in the respiratory tract included epithelial cell hyperplasia, squamous metaplasia, atrophy and accumulation of pigmented alveolar macrophages; they were mostly dose-dependent, more pronounced in the upper than lower respiratory tract and completely or partially reversed by 6 weeks post-inhalation. Qualitatively, the biological effects seen for the EHC and the 1R4F were comparable and similar to those observed in other mainstream smoke inhalation studies. Quantitatively, the biological activity of the EHC mainstream smoke was, on average, 65% lower than that of the 1R4F mainstream smoke on an equal cigarette basis and equivalent activity on an equal TPM basis.

Evaluation of the potential effects of ingredients added to cigarettes. Part 4: subchronic inhalation toxicity.

Mainstream smoke from blended research cigarettes with (test) and without (control) the addition of ingredients to the tobacco was assayed for inhalation toxicity. 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. Male and female Sprague-Dawley rats were exposed nose-only either to fresh air (sham) or diluted mainstream smoke from the test, the control, or the Reference Cigarette 1R4F at a concentration of 150 microg total particulate matter/l for 90 days, 6h/day, 7 days/week. A 42-day post-inhalation period was included to evaluate reversibility of possible findings. There were no remarkable differences in in-life observations or gross pathology between test and control groups. An increase in activity of liver enzymes, known to be due to the high smoke dose, revealed no toxicologically relevant differences between the test and control groups. No toxicological differences were seen between the test and control groups for smoke-related hematological changes, such as a decrease in total leukocyte count. The basic smoke-related histopathological effects, which were more pronounced in the upper respiratory tract than in the lower respiratory tract, were hyperplasia and squamous metaplasia of the respiratory epithelium, squamous metaplasia and atrophy of the olfactory epithelium, and accumulation of pigmented alveolar macrophages. There were no relevant qualitative or quantitative differences in findings in the respiratory tract of the rats exposed to the smoke from the control and test cigarettes. The data indicate that the addition of these 333 commonly used ingredients, added to cigarettes in three groups, did not increase the inhalation toxicity of the smoke, even at the exaggerated levels used.

Changes in the expression of alpha(2)-adrenergic receptor subtypes during maturation of neuronal cells from fetal pig superior cervical ganglia.

The expression of presynaptic alpha(2)-adrenergic receptor (alpha(2)-AR) subtypes was investigated in cultured neurons from fetal pig superior cervical ganglion (SCG). Cells were incubated with chicken antibodies against alpha(2)A-, alpha(2)B- or alpha(2)C-AR subtypes either alone or together with antibodies against dopamine-beta-hydroxylase (DbetaH, a marker for adrenergic neurons) or against choline acetyl transferase (ChAT, a marker for cholinergic neurons). We found immunoreactivity for all three alpha(2)-AR subtypes in SCG-cells when cultured for 8-11 days. The relative expression of the alpha(2)A-subtype was approximately 1/3 of that of alpha(2)B- and alpha(2)C-AR. Co-localisation of all three alpha(2)-AR subtypes was observed in cells expressing DbetaH or ChAT. Increasing the potassium concentration in the culture medium increased the expression of DbetaH and decreased the expression of the alpha(2)A- and alpha(2)C-subtype without altering the expression of the alpha(2)B-subtype. Co-culture of neurons with pig splenocytes enhanced the expression of ChAT and decreased the expression of the alpha(2)B-subtype without altering the expression of alpha(2)A- and alpha(2)C-subtypes. Our results indicate that the three alpha(2)-receptor subtypes are expressed on both noradrenergic and cholinergic nerves. Induction of the noradrenergic phenotype favours the expression of the alpha(2)B-subtype over that of the alpha(2)A- and alpha(2)C-subtype. Conversely, enhancement of the cholinergic phenotype favours the expression of the alpha(2)A- and alpha(2)C-subtypes over that of the alpha(2)B-subtype. Our results suggest that the alpha(2)B-receptor is preferentially associated with noradrenergic nerve endings.

Localization of alpha 2-adrenergic receptor subtypes in the anterior segment of the human eye with selective antibodies.

PURPOSE: To develop antibodies that selectively recognize each of the alpha 2-adrenergic receptor (AR) subtypes and to determine the expression and localization of these subtypes in the anterior segment of the human eye. METHODS: Recent studies have shown that there are three subtypes of the alpha 2-ARs, termed alpha 2-C10 (alpha 2A), alpha 2-C2 (alpha 2B), and alpha 2-C4 (alpha 2C). Polymerase chain reaction was used to amplify portions of these receptors fused (in-frame) to a cDNA encoding glutathione-S-transferase (GST). The expressed fusion proteins were used to immunize chickens, and antibodies were generated. Immunofluorescence microscopy was used to localize the alpha 2-AR subtypes in sections of human and rabbit ciliary body. Polymerase chain reaction and dot blot hybridization were used to determine which subtypes were present in RNA from primary cultures of human nonpigmented epithelium (NPE) and rabbit iris-ciliary body (ICB). RESULTS: Immunofluorescence microscopy of COS cells transfected with the alpha 2-AR subtypes showed that the antibodies raised against the GST-receptor fusion proteins specifically recognized their respective receptor subtypes. In the human ciliary body, alpha 2 B and alpha 2C immunoreactivity were present in the NPE and ciliary muscle. In the rabbit ciliary body, alpha 2A immunoreactivity also was present. Polymerase chain reaction and dot blot hybridization indicated that RNA encoding the alpha 2B and alpha 2C subtypes was present in human NPE and that RNA encoding all three subtypes was present in the rabbit ICB. CONCLUSIONS: Multiple alpha 2-adrenergic subtypes are expressed in the ciliary body. In the human, alpha 2B and alpha 2C predominate, whereas all three are present in the rabbit. This could be important with respect to animal models of glaucoma and to the development of drugs for lowering intraocular pressure.

Expression of type I and type IB receptors for activin in midgestation mouse embryos suggests distinct functions in organogenesis.

Activins exert their effects by inducing heteromeric complexes of either of two different type I receptors, ActR-I or ActR-IB, and either of two type II receptors, ActR-II or ActR-IIB. We describe the cDNA cloning of the mouse homologue of human ActR-IB and analyze binding of radio-iodinated activin on type I/type II combinations of mouse receptors expressed from cDNA. We studied the distribution of ActR-I and ActR-IB mRNAs in postimplantation mouse embryos by in situ hybridization. In the 12.5-day postcoitum embryo, both mRNAs are found in the brain, spinal cord, some ganglia, vibrissae, lungs, body wall, stomach, gonads, ribs, limbs and shoulders. ActR-I mRNA, but not ActR-IB, is expressed in blood vessels, the heart, tongue, intervertebral discs and diaphragm. Conversely, only ActR-IB mRNA is detected in the olfactory region, eye, tooth primordium, esophagus, mesonephros, dorsal root ganglia and is strongly expressed in the spinal cord. Our results demonstrate similarities, but also differences and complementarities (mesenchymal versus epithelial expression) between the expression patterns of these type I receptors. Moreover, their expression patterns overlap with at least one of the type II activin receptors and/or one of activin subunits in some regions of the embryo, such as the brain, spinal cord, pituitary, whisker follicles, and the inner nuclear neuroblastic layer of the eye.

Thermodynamic analysis of isoproterenol binding to beta-adrenoceptors in rat lung membranes.

The thermodynamic properties of the binding of the beta-adrenoceptor agonist isoproterenol and of the antagonist propranolol to beta-adrenoceptors of rat lung were investigated. We found that in our experimental conditions, the high- and low-affinity binding sites for the agonist displayed different properties: the binding to the high-affinity binding site was entropy-driven with a small increase in enthalpy, while agonist binding to the low-affinity binding site was enthalpy-driven. Binding of isoproterenol in the presence of GTP or its non-hydrolyzable analogue GppNHp, and the binding of propranolol were enthalpy-driven with a small increase in entropy.

Antibodies to a human alpha 2-C10 adrenergic receptor fusion protein confirm the cytoplasmic orientation of the V-VI loop.

DNA encoding the hydrophilic region between transmembrane domains V and VI of the human platelet alpha 2-adrenergic receptor (alpha 2-C10) was amplified using the polymerase chain reaction and was cloned in-frame with a portion of the gene encoding glutathione-S-transferase (GST). Expression of the recombinant plasmid in E. coli resulted in the production of a GST/alpha 2-C10 fusion protein which was purified by preparative SDS-PAGE. Chickens inoculated with the fusion protein produced antibodies that were present in their eggs. In cells expressing the alpha 2-C10, these antibodies recognized the receptor in both Western Blots and indirect immunofluorescence. For the immunofluorescence studies, antibody recognition required permeabilization of the cells with detergent. This evidence establishes the cytoplasmic orientation of the V-VI loop and supports the general model for G-protein coupled receptors.

Influence of aging on fluidity and coupling between beta-receptors and G-proteins in rat lung membranes.

We tested the hypothesis that in rat lung membranes, the age-related decline in the percentage of beta-receptors coupled with high affinity to G-proteins, is due to limitation of the diffusion caused by a decrease in membrane fluidity. We measured both parameters simultaneously in a crude membrane preparation from lungs of rat of different age. In contrast to what is found in crude membrane preparations from rat liver and brain, in rat lung fluidity was increased upon aging. We conclude that the age-related alteration in coupling between receptor and G-protein is difficult to explain by alterations of membrane fluidity.

Pharmacological characterization of a beta 3-receptor agonist (BRL 37,344) and a partial agonist (CGP 12,177A) in neonatal rat liver plasma membranes.

The pharmacological properties of BRL 37,344 (sodium-4-(2'-[2-hydroxy-2- (3-chloro-phenyl)ethylamino]-propyl)phenoxyacetatesesquihydrate), a beta 3-selective agonist, and CGP 12,177A) (-)-4-(3-t-butyl amino-2-hydroxypropoxy) benzimidazole-2-one], a non-selective beta-antagonist, recently characterized as a partial beta 3-agonist in rat adipose tissue, were studied in comparison with isoproterenol, a non-selective beta-agonist, in plasma membranes prepared from the livers of newborn rats. Competition binding curves obtained with [125I]iodocyanopindolol ([125I]CYP) as ligand and isoproterenol or BRL 37,344 as competitor were characterized by the presence of a high and a low affinity binding site; the high affinity binding site was no longer detectable when guanidylimidobisphosphate (GppNHp) was present in the incubation mixture. Competition curves with CGP 12,177A were monophasic and independent of GppNHp. In the presence of 10(-7) M of the beta 2-selective antagonist ICI 118,551 [erythro-(+/-)-1-(7-methylindan-4-yloxy)-3-isopropylamino butan-2-ol], a concentration which blocks most of the beta 2-receptors, ligand binding was reduced to 32% of its maximum. Under these conditions, isoproterenol further displaced the ligand, and competition curves still displayed the high and the low affinity binding sites; BRL 37,344, however, caused no further displacement of ligand, except at the highest concentrations. This suggests that BRL 37,344 occupies only the ICI 118,551-sensitive binding sites, i.e. beta 2-receptors. Isoproterenol and BRL 37,344 both stimulated adenylate cyclase (EC 4.6.1.1) activity concentration dependently, although the stimulating effect of BRL 37,344 was about half of what was found for isoproterenol. Furthermore, BRL 37,344 inhibited concentration dependently the isoproterenol-induced stimulation of adenylate cyclase, and the inhibition was dependent on the concentration of isoproterenol. The stimulating effect of isoproterenol and BRL 37,344 on adenylate cyclase was blocked by ICI 118,551, whereas the beta 1-selective antagonist CGP 20,712A ((+/-)-(2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino)-3-[4-(1-methy l-4- trifluoromethyl-2-imidazolyl)-phenoxy]-2-propanolmethane sulphonate) was ineffective. CGP 12,177A failed to stimulate adenylate cyclase activity. From these results we suggest that BRL 37,344 acts as a beta 2-partial agonist in rat liver. The results obtained with CGP 12,177A are typical for a non-selective beta-antagonist. We therefore conclude that there is no pharmacological evidence for the presence of beta 3-receptors in livers from newborn rats.

Influence of age on the beta 1- and beta 2-adrenergic receptors in rat liver.

The influence of maturation and aging on beta receptors in rat liver was studied. Competition binding experiments with the nonselective beta-antagonist propranolol and the subtype selective antagonists ICI 118,551 (beta 2) ICI 89,406 (beta 1), and CGP 20,712A (beta 1) revealed the presence of a mixed beta 1 and beta 2 receptor population in crude plasma membrane preparations from livers of newborn, mature, and senescent rats. The percentage of beta 1 receptors was lowest in livers from newborn rats and was increased in livers from mature and senescent rats. This increase is caused by a decrease in beta 2 receptor density on maturation, although the beta 1 receptor density is nearly constant throughout the life span of the rat. Isoproterenol-stimulated adenylate cyclase activity was inhibited in livers from senescent rats by propranolol and ICI 118,551 and to a lesser extent by ICI 89,406 and CGP 20,712A. The isoproterenol-stimulated glucose output in hepatocytes from senescent rats was inhibited concentration dependently by propranolol, ICI 118,551, ICi 89,406, and CGP 20,712A. From these results we conclude that beta 1 and beta 2 receptors are present in livers from rats of the three age groups and that the beta 1 to beta 2 receptor ratio is increased in livers from mature and senescent rats compared with newborn rats. Both beta receptor subtypes are linked to the cAMP second messenger system in newborn and senescent rats; beta 1 and beta 2 receptors are equally involved in the regulation of glycogenolysis in hepatocytes from senescent rats.

Effect of age on the kinetics of the binding of iodocyanopindolol to a membrane preparation of rat lungs.

The association (k+1) and dissociation (k-1) rate constants, and the equilibrium thermodynamic binding parameters (delta G0, delta H0 and delta S0) of the beta-adrenergic ligand [125Iodo]cyanopindolol (ICYP) were studied in a crude lung membrane preparation of rats of different ages. There was no difference in k+1-values for the different age groups, while the k-1-values were in all cases difficult to measure: almost no dissociation of ICYP from its binding site occurs. The thermodynamic properties were not affected by age. It is concluded that, in these experimental conditions, age has no effect on the kinetic parameters of the binding of ICYP to the beta-adrenoceptors in rat lung.

Characterization of the beta-adrenergic transduction system in spleen mononuclear leukocyte membranes of young and senescent rats.

The effect of aging on some of the properties of the beta-adrenergic transduction system was determined in a membrane fraction of spleen mononuclear leukocytes from young (2-3 months) and old (24-25 months) rats. Receptor density was unchanged and the percentage of receptors in the high affinity configuration for isoproterenol was reduced with increasing age. Adenylate cyclase activity, either unstimulated or stimulated with forskolin, GTP or isoproterenol was not affected by aging. This suggests the presence of compensatory mechanisms in the beta-adrenergic signal transduction system of rat spleen mononuclear cells in order to ensure equal cAMP production for equal agonist stimulation.

The beta-adrenergic transduction system in kidneys from young and senescent rats.

beta-Adrenoceptor density, ligand affinity, high-affinity agonist binding, basal adenylate cyclase activity and cAMP synthesis upon stimulation with either forskolin, F-, guanine nucleotides (GTP or GppNHp) or isoproterenol in the presence of the nucleotides were studied in membranes prepared from kidneys of young (2-3 month) and old (24-25 month) male Wistar rats. There is a significant (P less than 0.01) 62% increase in beta-receptor density, a significant (P less than 0.05) 115% decrease in ligand affinity, a significant (P less than 0.05) 33% decrease of high-affinity binding sites for (-)-isoproterenol and a significant (P less than 0.01) 151% decrease of the affinity of the high-affinity agonist binding site. Basal adenylate cyclase activity and the activity after stimulation with guanine nucleotides and forskolin were significantly higher in old animals as compared to young (P less than 0.01). Stimulation of the system with isoproterenol in the presence of GTP was more effective in old animals, although the P less than 0.05 level of significance was barely reached. It is suggested that age-dependent changes of the beta-adrenoceptors in rat kidney are similar to those described for lungs: changes at the different levels of the beta-adrenergic transduction chain associated with age are compensatory so as to ensure equal cAMP synthesis for a given agonist stimulation.

Effect of aging on properties and function of beta-adrenoceptors in rat lung.

beta-Adrenoceptor density and ligand affinity, basal adenylate cyclase activity and cAMP synthesis upon stimulation with forskolin, fluoride, guanine nucleotides (GTP or guanylyl imidodiphosphate (GppNHp) or isoproterenol in the presence of the nucleotides were studied in membranes prepared from lungs of young (aged 2-3 months) and of old (aged 24-25 months) male Wistar rats. There was a significant (P less than 0.05, 21%) increase in beta-receptor density and a significant (P less than 0.05, 38%) decrease in the percentage of high-affinity binding sites for isoproterenol. Both basal adenylate cyclase activity and that after stimulation with guanine nucleotides or isoproterenol in the presence of nucleotides were unaltered with age. Forskolin stimulation of cAMP synthesis was significantly reduced (by 24%, P less than 0.05) in tissues from older animals. It is suggested that the age-dependent changes in properties of beta-receptors in rat lungs are compensatory, in order to ensure equal cAMP production for equal agonist stimulation.

Evaluation of a semiautomatic cell harvester filtration for the determination of beta-adrenoceptors in human mononuclear leukocytes.

The ligand affinity (Kd) and the number (Bmax) of beta-adrenoceptors in human mononuclear leukocytes was measured using a semiautomatic cell harvester method for the separation of bound and free ligand. The method was first evaluated in terms of contamination, nonspecific filter binding, and well-to-well reproducibility. A comparison was then made with the conventional manual filtration method. The Kd values for the binding of [125lodo]-cyanopindolol to mononuclear leukocyte membranes were independent of the method, whereas the Bmax values were systematically higher (around 30%) when determined with the cell harvester filtration method. A highly significant correlation was found between the Bmax values obtained with both methods.