Comparative assessment of HPHC yields in the Tobacco Heating System THS2.2 and commercial cigarettes.

There has been a sustained effort in recent years to develop products with the potential to present less risk compared with continued smoking as an alternative for adult smokers who would otherwise continue to smoke cigarettes. During the non-clinical assessment phase of such products, the chemical composition and toxicity of their aerosols are frequently compared to the chemical composition and toxicity of the smoke from a standard research cigarette - the 3R4F reference cigarette. In the present study, it is demonstrated that results of these analytical comparisons are similar when considering commercially available cigarette products worldwide. A market mean reduction of about 90% is observed on average across a broad range of harmful and potentially harmful constituents (HPHC) measured in the aerosol of a candidate modified risk tobacco product, the Tobacco Heating System 2.2 (THS2.2), compared against the levels of HPHC of cigarettes representative of selected markets; this mean reduction is well in line with the reduction observed against 3R4F smoke constituents in previous studies.

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.

Selective pressure during tumor promotion by phenobarbital leads to clonal outgrowth of beta-catenin-mutated mouse liver tumors.

Tumor promoters are non-mutagenic chemicals which increase the probability of cancer by accelerating the clonal expansion of cells transformed during tumor initiation. Phenobarbital (PB) is an antiepileptic drug which promotes hepatocarcinogenesis in rodents when administered subsequent to an initiating carcinogen like diethylnitrosamine (DEN). Here we have investigated the prevalence and patterns of mutations in two genes, Ha-ras and beta-catenin, both known mutational targets in mouse hepatocarcinogenesis. Liver tumors were generated by a single administration of DEN to 6 week old mice followed by feeding of PB (0.05%) containing or control diet for 39 weeks. Mutations at Ha-ras codon 61 were screened by allele-specific oligonucleotide hybridization; beta-catenin mutations were detected by direct sequencing of PCR products spanning exon 2. In tumors from mice treated with DEN alone, the prevalence of Ha-ras mutations was approximately 30% (6/20), while no beta-catenin mutations (0/13) were detectable in tumors of this treatment group. By contrast, Ha-ras mutations were undetectable in tumors from mice treated with DEN/PB (0/32), while approximately 80% (37/46) of tumors from this group showed beta-catenin mutations. These results demonstrate that PB strongly affects the prevalence of mutations in the two cancer-related genes, presumably by positive and negative selection for cells harboring the respective mutation.

Lack of phenobarbital-mediated promotion of hepatocarcinogenesis in connexin32-null mice.

Connexin32 (Cx32) is the major gap junction forming protein in liver. We have recently shown that hepatocarcinogenesis is strongly enhanced in mice deficient in Cx32, demonstrating that lack of functional Cx32 accelerates liver tumorigenesis. Many tumor-promoting agents, including phenobarbital, block gap junctional intercellular communication in vitro, and it has been suggested that this effect is relevant for clonal expansion of neoplastic cells in vivo. We have now tested this hypothesis by analyzing the potency of phenobarbital as a liver tumor promoter in male Cx32-wild-type (Cx32(Y/+)) and Cx32-null (Cx32(Y/-)) mice. Preneoplastic and neoplastic liver lesions were induced in 6-week-old male mice by a single injection of 90 microg/g body weight of N-nitrosodiethylamine, and groups of mice were subsequently kept on phenobarbital-containing (0.05%) or control diet for 39 weeks. Frozen liver sections were prepared, and (pre)neoplastic lesions were identified by their deficiency in glucose-6-phosphatase staining. In addition, the number and size of macroscopically visible tumors were monitored. Phenobarbital led to a approximately 5-fold increase in the volume fraction occupied by glucose-6-phosphatase-deficient liver lesions in Cx32(Y/+) mice, whereas there was no such increase in Cx32(Y/-) mice. Even more pronounced differences were observed with respect to tumor response. Whereas phenobarbital clearly promoted the occurrence of numerous large hepatomas in Cx32(Y/+) mice, no such effect was seen in Cx32(Y/-) mice. These results demonstrate, for the first time, that functional Cx32 protein is required for tumor promotion by phenobarbital.

Hepatocarcinogenesis in female mice with mosaic expression of connexin32.

Mice deficient for connexin32 (Cx32), the major gap junction forming protein in liver, are highly susceptible to hepatocarcinogenesis. Because the Cx32 gene is located on the X-chromosome, heterozygous females show mosaicism with respect to Cx32 expression; this enables their use in studying the effect of Cx32-deficiency in a mixed Cx32-plus/Cx32-minus environment in vivo. Female C3H/He mice (Cx32(+/+)) were crossed with Cx32-deficient C57BL/129Sv males (Cx32(Y/-)) to yield F1 females heterozygous with respect to Cx32 (Cx32(+/-)). Patches of hepatocytes were observed in normal liver that either expressed Cx32 or failed to express the protein. The mean fraction of Cx32-negative tissue in liver was about 60% and did not change significantly with age of mice. Neoplastic liver lesions, induced in weanling mice, were identified in serial liver sections by their deficiency in glucose-6-phosphatase staining. Parallel sections were used for immunohistochemical demonstration of Cx32 protein. Smaller lesions were either homogenously Cx32-negative or showed unchanged to slightly elevated levels of Cx32 protein. There were no major differences in number and size distribution between lesions of these 2 phenotypes. In addition, larger lesions were mostly Cx32-negative but often contained embedded patches of Cx32-positive cells. Staining for the proliferation-associated nuclear antigen Ki-67 did not reveal significant differences between Cx32-negative and Cx32-positive hepatocytes in Cx32-mosaic tumors. This suggests that expression of Cx32 within a subpopulation of tumor cells does not negatively regulate their growth nor does it seem to affect the proliferation of their directly neighboring Cx32-negative counterparts.

The effect of connexin32 null mutation on hepatocarcinogenesis in different mouse strains.

Connexin32 (Cx32) is the major gap junctional protein in mouse liver. We have shown recently that the formation of liver tumours in Cx32-deficient mice is strongly increased in comparison with control wild-type mice, demonstrating that the deficiency in gap junctional communication has an enhancing effect on hepatocarcinogenesis. We have now compared the effect of Cx32 deficiency on liver carcinogenesis in two strains of mice with differing susceptibility to hepatocarcinogenesis. Heterozygous Cx32(+/-) females were crossed with male Cx32 wild-type C57BL/6J (low susceptibility) or C3H/He (high susceptibility) mice. Since the Cx32 gene is located on the X-chromosome, the resulting F1 males segregated to the genotypes Cx32(Y/+) and Cx32(Y/-). Genotyping was performed by PCR-analysis using tail-tip DNA. Weanling male mice were i.p. injected with a single dose of N-nitrosodiethylamine and were killed 16, 21 or 26 weeks later. The number, volume fraction and size distribution of precancerous liver lesions characterized by a deficiency in the marker enzyme glucose-6-phosphatase were quantitated. The results demonstrate that Cx32 deficiency only slightly affects the number of enzyme-altered lesions, but strongly enhances their growth, both in the resistant and the susceptible mouse strain, suggesting that decreased intercellular communication results in tumour promoting activity irrespective of the genetic background of the mouse strain used. Since Cx32-deficient C3H/He hybrids were approximately 5-10 times more sensitive than C3H/He hybrids with an intact Cx32 gene, this mouse strain may prove very useful for toxicological screening purposes.