Naoki Kunugita

GENETIC POLYMORPHISMS OF TOBACCO- AND ALCOHOL-RELATED METABOLIZING ENZYMES AND ORAL CAVITY CANCER

Both genetic and environmental factors are involved in the development of cancer. Oral cavity cancer has been reported to be epidemiologically associated with tobacco and alcohol consumption. We examined genetic polymorphisms of the glutathione‐S‐transferase (GST) M1/T1, cytochrome P‐450 (CYP) 1A1/2E1 and aldehyde dehydrogenase 2 (ALDH2) genes in 92 Japanese patients with oral cavity cancer and 147 unrelated non‐cancer Japanese controls. There was a significant association between cigarette smoking and cancer risk but no significant association between alcohol consumption and cancer risk. The frequency of the GSTM1 null genotype was significantly higher in cancers (58.7%) compared with controls (46.3%). However, there were no significant differences between controls and patients with oral cavity cancer in the polymorphisms of the GSTT1, CYP1A1, CYP2E1 and ALDH2 genes. From statistical evaluation on various combinations of genotypes, we did not observe any gene combinations associated with cancer risk. There were also no genetic polymorphisms associated with increased risk of oral cavity cancer among smokers and drinkers. These results imply that the GSTM1 null genotype has a weak correlation, but another 4 genetic polymorphisms are unlikely to be associated, with oral cavity cancer among Japanese. Int. J. Cancer 83:606–609, 1999.

Aldehyde dehydrogenase (ALDH) 2 associates with oxidation of methoxyacetaldehyde; in vitro analysis with liver subcellular fraction derived from human and Aldh2 gene targeting mouse.

A principal pathway of 2‐methoxyethanol (ME) metabolism is to the toxic oxidative product, methoxyacetaldehyde (MALD). To assess the role of aldehyde dehydrogenase (ALDH) in MALD metabolism, in vitro MALD oxidation was examined with liver subcellular fractions from Japanese subjects who carried three different ALDH2 genotypes and Aldh2 knockout mice, which were generated in this study. The activity was distributed in mitochondrial fractions of ALDH2*1/*1 and wild type (Aldh2+/+) mice but not ALDH2*1/*2, *2/*2 subjects or Aldh2 homozygous mutant (Aldh2−/−) mice. These data suggest that ALDH2 is a key enzyme for MALD oxidation and ME susceptibility may be influenced by the ALDH2 genotype.

Carbonyl Compounds Generated from Electronic Cigarettes

Electronic cigarettes (e-cigarettes) are advertised as being safer than tobacco cigarettes products as the chemical compounds inhaled from e-cigarettes are believed to be fewer and less toxic than those from tobacco cigarettes. Therefore, continuous careful monitoring and risk management of e-cigarettes should be implemented, with the aim of protecting and promoting public health worldwide. Moreover, basic scientific data are required for the regulation of e-cigarette. To date, there have been reports of many hazardous chemical compounds generated from e-cigarettes, particularly carbonyl compounds such as formaldehyde, acetaldehyde, acrolein, and glyoxal, which are often found in e-cigarette aerosols. These carbonyl compounds are incidentally generated by the oxidation of e-liquid (liquid in e-cigarette; glycerol and glycols) when the liquid comes in contact with the heated nichrome wire. The compositions and concentrations of these compounds vary depending on the type of e-liquid and the battery voltage. In some cases, extremely high concentrations of these carbonyl compounds are generated, and may contribute to various health effects. Suppliers, risk management organizations, and users of e-cigarettes should be aware of this phenomenon.

Measurement of the Physical Properties of Aerosols in a Fullerene Factory for Inhalation Exposure Assessment

Assessment of human exposure is important for the elucidation of potential health risks. However, there is little information available on particle number concentrations and number size distributions, including those of nanoparticles, in the working environments of factories producing engineered nanomaterials. The authors used a scanning mobility particle sizer and an optical particle counter to measure the particle number size distributions of particles ranging in diameter (D p ) from 10 nm to >5000 nm in a fullerene factory and used scanning electron microscopy to examine the morphology of the particles. Comparisons of particle size distributions and morphology during non-work periods, during work periods, during an agitation process, and in the nearby outdoor air were conducted to identify the sources of the particles and to determine their physical properties. A modal diameter of 25 nm was found in the working area during the non-work period; this result was probably influenced by ingress of outdoor air. During the removal of fullerenes from a storage tank for bagging and/or weighing, the particle number concentration at D p <50 nm was no greater than that in the non-work period, but the concentration at D p >1000 nm was greater during the non-work period. When a vacuum cleaner was in use, the particle number concentration at D p <50 nm was greater than that during the non-work period, but the concentration at D p >1000 nm was no greater. Scanning electron microscopy revealed that the coarse particles emitted during bagging and/or weighing were aggregates/agglomerates of fullerenes; although origin of particles with D p <50 nm is unclear.

Expression of cytochrome P450 in tumor tissues and its association with cancer development.

CYPs (cytochrome P450s) catalyze the conversion of numerous numbers of xenobiotics including carcinogens and drugs. CYPs can be involved in metabolic pathways of activation of procarcinogens and/or inactivation of carcinogens during the tumorigenic processes. Recently, increasing number of cancer tissues as well as normal tissues have been found to express a variety of CYPs. The local expression of CYPs in tumors appears to be very important for the management of cancers since CYPs expressed in tumors may be involved in activation and/or inactivation of anticancer drugs. The expression of CYPs in tumors may also convert endogenous substrates to metabolites that facilitate cancer development. In this review, we summarize the association of CYP expression in cancer tissues with carcinogenesis and cancer treatment.

Determination of acrolein and other carbonyls in cigarette smoke using coupled silica cartridges impregnated with hydroquinone and 2,4-dinitrophenylhydrazine.

A new method for the determination of acrolein and other carbonyls in cigarette smoke using a dual cartridge system has been developed. Each cartridge consists of reagent-impregnated silica particles. The first contains hydroquinone (HQ) for the inhibition of acrolein polymerization, while the second contains 2,4-dinitrophenylhydrazine (DNPH) for the derivatization of carbonyls. Smoke samples were firstly drawn through the cartridge containing HQ-impregnated silica (HQ-silica) and then through the DNPH-impregnated silica (DNPH-silica). Acrolein in the sample was completely trapped in the first HQ-silica cartridge. Some other airborne carbonyls were also trapped by the HQ-silica, and those that pass through were trapped in the second DNPH-silica cartridge. Extraction was performed in the reverse direction to air sampling. When solvent was eluted through the dual-cartridges, excess DNPH was washed into the HQ bed where it reacted with acrolein and other trapped carbonyls to form the corresponding hydrazone derivatives. All of the hydrazones derived from airborne carbonyls were completely separated and measured using high-performance liquid chromatography. This HQ-DNPH-method can be applied for the determination of acrolein and other alpha,beta-unsaturated aldehydes, such as crotonaldehyde, in cigarette smoke.

Climbing exercise enhances osteoblast differentiation and inhibits adipogenic differentiation with high expression of PTH/PTHrP receptor in bone marrow cells

We developed previously a mouse voluntary climbing exercise model as a physiological mechanical loading model and reported that climbing exercise increased bone formation, but its effect on adipogenesis is unknown. We assessed the effects of loading and PTH/PTHrP receptor (PTHR1) on bone marrow adipocyte differentiation in relation with osteoblast differentiation. 8-week-old C57BL/6J male mice were divided into ground control (GC) and climbing exercise (EX) group. Mice were housed in 100-cm towers and climbed up toward a bottle placed at the top of the cage to drink water. The values of bone volume and osteoblast number were significantly higher while those of marrow adipocyte volume and number were significantly lower in the 28dayEX group than 28dayGC group. The mRNA expression levels of adipocyte differentiation genes CCAAT/enhancer-binding proteins (C/EBP) beta and delta were lower in 4dayEX mice, while the adipocyte specific genes fatty acid binding protein (aP2) and phosphoenolpyruvate carboxykinase (PEPCK) expressions were lower in 7dayEX mice. In primary bone marrow cell cultures, the number of alkaline phosphatase-positive colony forming units-fibroblastic (ALP+ CFU-f) and Oil-red-O-positive cells were both increased in the 4dayEX group. Climbing exercise transiently increases both osteogenic and adipogenic potential in bone marrow stromal cells, and inhibits terminal adipocyte differentiation and promotes osteoblast differentiation. Immunoreactivity for the PTHR1 was intense on osteoblastic cell lineage in the endosteal tibial metaphysis. PTHR1 mRNA expression was increased in 4dayEX mice and PTHR1-positive cells were increased after 7 days in the experimental group. Ex vivo addition of PTHR1 antibody decreased and that of PTHrP(1-34) increased the number of ALP+ CFU-f in bone marrow cell cultures obtained at 4 days after the exercise, while the addition of PTHR1 antibody increased and PTHrP(1-34) decreased the number of Oil-red-O-positive cells. Our results indicate that climbing exercise enhanced osteoblast differentiation and inhibited terminal differentiation of adipocyte progenitors with high expression of PTHR1 in bone marrow cells.

Derivatization of carbonyl compounds with 2,4-dinitrophenylhydrazine and their subsequent determination by high-performance liquid chromatography ☆

Derivatization of carbonyl compounds with 2,4-dinitrophenylhydrazine (DNPH) is one of the most widely used analytical methods. In this article, we highlight recent advances using DNPH provided by our studies over past seven years. DNPH reacts with carbonyls to form corresponding stable 2,4-DNPhydrazone derivatives (DNPhydrazones). This method may result in analytical error because DNPhydrazones have both E- and Z-stereoisomers caused by the CN double bond. Purified aldehyde-2,4-DNPhydrazone demonstrated only the E-isomer, but under UV irradiation and the addition of acid, both E- and Z-isomers were seen. In order to resolve the isometric problem, a method for transforming the CN double bond of carbonyl-2,4-DNPhydrazone into a C-N single bond, by reductive amination using 2-picoline borane, has been developed. The amination reactions of C1-C10 aldehyde DNPhydrazones are completely converted into the reduced forms and can be analyzed with high-performance liquid chromatography. As a new application using DNPH derivatization, the simultaneous measurement of carbonyls with carboxylic acids or ozone is described in this review.

Disruption of the p53 gene results in preserved trabecular bone mass and bone formation after mechanical unloading.

We tested the hypothesis that mechanical unloading facilitates signaling of p53, an important modulator of cell cycling and apoptosis, in bone marrow cells and thereby reduces trabecular bone volume (BV). We performed histomorphometric analyses and bone marrow cell cultures in tail‐suspended (TS) p53 null (p53−/−) and wild‐type (p53+/+) mice. Eight‐week‐old male mice were assigned to four groups after 1‐week acclimatization: p53+/+ + ground control (GC), p53+/++TS, p53−/−+GC, and p53−/−+TS. Bilateral tibial samples were used for analysis. The histomorphometric parameters of trabecular structure, formation and resorption did not differ between the p53−/−+GC and p53+/++GC groups. Trabecular BV in p53+/++TS mice was significantly reduced to 45% of that in the p53+/++GC group after one week of TS. In contrast, BV in p53−/−+TS mice was preserved at the same level as that in the p53−/−+GC group. The bone formation rate (BFR) was significantly reduced in p53+/++TS but not in p53−/−+TS mice. Unloading significantly increased trabecular osteoclast number (Oc.N) and surface in p53+/++TS mice compared with the p53+/++GC group, but the difference was not significant between p53−/−+TS and p53−/−+GC mice. In bone marrow cell culture, the numbers of alkaline phosphatase‐positive (ALP+) colony‐forming units fibroblastic (CFU‐f) and mineralized nodules were significantly reduced in p53+/++TS, but not p53−/−+TS mice. [3H]thymidine incorporation into bone marrow cells was higher in p53−/− mice than in p53+/+ mice, independent of mechanical loading or unloading. Flow cytometric cell cycle analysis revealed that unloading significantly increased the percentage of hypoploid bone marrow cells in p53+/+ mice relative to that in p53+/++GC mice, but there was no significant difference in ploidy between p53−/−+TS and p53−/−+GC mice. Expression levels of p53 and p21 mRNAs were enhanced after TS in bone marrow cells from p53+/+ mice. Our data show that trabecular bone mass and bone formation were preserved after tail‐suspension in p53−/− mice, closely associated with ALP+ CFU‐f and mineralized nodule formation in marrow cultures obtained from tibias of p53−/− mice. We speculate that bone loss due to mechanical unloading may be related to facilitation of intracellular p53‐p21 signaling.

Effect of prolonged exposure to low concentrations of formaldehyde on the corticotropin releasing hormone neurons in the hypothalamus and adrenocorticotropic hormone cells in the pituitary gland in female mice.

We examine the effect on the hypothalamus-pituitary-adrenal gland (HPA) axis of prolonged exposure to low levels of formaldehyde in female C3H/He mice, using immunocytochemical and RT-PCR methods. Two groups of female mice were exposed to differing concentrations (0, 80, 400, 2000 ppb) of formaldehyde inhalation for 16 h/day, 5 days/week, for 12 weeks. The corticotropin releasing hormone (CRH)-immunoreactive (ir) neurons in the hypothalamus were then examined, together with the adrenocorticotropin hormone (ACTH)-ir cells and ACTH mRNA in the pituitary. One group comprised sham control mice. The other group was made allergic by injection of ovalbumin (OVA) and alum prior to exposure to formaldehyde, since most sick building syndrome (SBS) sufferers are women with allergic disease. These animals were further exposed to aerosolized OVA as a booster four times during the exposure period. Our results showed a dose-dependent increase in the number of CRH-ir neurons in the non-allergy (NAG) group. A similar pattern was found in ACTH-ir cells and ACTH mRNA. The allergy (AG) model group showed an increase in basal levels of all markers of HPA activity. Moreover, the AG mice appeared to respond to the lowest concentration of formaldehyde, and all indices of HPA activity were reduced at the highest concentrations of formaldehyde. These results relate to an important clinical issue and also have implications in the broader area of HPA regulation. We conclude that our experimental system may be a suitable animal model for SBS and/or multiple chemical sensitivity (MCS).