In silico analysis of interaction pattern switching in ligandreceptor binding in Golgi α-mannosidase II induced by the protonated states of inhibitors.

Golgi α-mannosidase II (GM) is a pharmaceutical target for the design of inhibitors with anticancer activity. The known potent GM inhibitors undergo complex interactions with Zn2+ ions and the active-site amino acids, many of which contain ionisable functional groups. Herein, the physical insight into the ligandreceptor interactions has been provided based on energy decomposition techniques: SAPT (symmetry adapted perturbation theory) and FMO-PIEDA (fragment molecular orbital-pair interaction energy decomposition analysis) for a large GM active-site cluster. Protonation-dependent molecular recognition in Golgi α-mannosidase was demonstrated for five inhibitors, mannose, and its transition state. Zn2+ ion and Asp472 induce the key interactions with the deprotonated inhibitors (bearing an amino group in the neutral state), followed by Asp92 and Asp341. This interaction pattern is consistent for all the studied inhibitors and is similar to the interaction pattern of the enzyme native substrate - mannose. The interactions with Zn2+ ion become repulsive for the protonated states of the inhibitors (bearing an amino group with +1 charge) and the mannosyl transition state. The importance of Asp92 and Asp204 considerably increases, while the interactions with Asp472 and Asp341 are slightly modified. The interaction pattern for the protonated ligands seems to have an oxocarbenium transition state-like character, rather than a Michaelis complex of GM. The electrostatic interactions with amino acids coordinating zinc ion are of key importance for both the neutral and protonated states of the inhibitors. The ligand's diol group has a dual role as an electron donor, coordinating zinc ion, and as an electron acceptor, interacting with Asp92 and Asp472 via strong hydrogen bonds. This interaction pattern is an essential structural feature of the potent GM inhibitors, which is consistent with the experimental findings. Based on the calculations, either the protonated or deprotonated state of the ligand may be the active form of the GM inhibitor, exhibiting different interacting patterns.

Anatomical location of various condylar points for jaw movement analysis in Japanese women.

The aim of this study was to determine anatomical locations of the hinge axis point, kinematic axis point and reference point for the palpated lateral condylar pole on lateral cephalograms. Subjects comprised 18 Japanese women selected according to following criteria: normal occlusion; and absence of signs and symptoms of stomatognathic function. Jaw movement and the condylar reference points noted earlier were recorded three-dimensionally with six degrees of freedom, and kinematic axis point and hinge axis point were determined using an optoelectronic jaw-tracking system. Lateral cephalograms were used to determine anatomical locations of the three points in the condyle. Mean location of hinge axis point was 12.9 mm anterior of the porion and 5.3 mm inferior to the Frankfort horizontal plane, the kinematic axis point was situated in 12.8 mm anterior and 0.1 mm inferior, and the reference point for the palpated lateral condylar pole was situated 10.7 mm anterior and 0.8 mm inferior, respectively. The kinematic axis point was located outside the condyle in the majority of subjects. The reference point for the palpated lateral pole offers a useful indicator in the analysis of condylar movements.

Correlation between facial morphology, mouth opening ability, and condylar movement during opening-closing jaw movements in female adults with normal occlusion.

The aim of this study was to investigate the relationship between parameters of facial morphology, maximal voluntary mouth opening ability, and condylar movements in 21 adult females, aged between 20 and 24 years. The subjects had a normal occlusion without sign or symptoms of temporomandibular joint (TMJ) dysfunction. Mandibular movements were recorded using an opto-electric jaw movement recording system with six degrees of freedom under a series of maximal mouth opening-closing movements. Maximal jaw opening and coincident condylar movement were measured three-dimensionally. The mean values of the incisor and condylar path were 41.1 +/- 3.5 mm (range 35.6-50.9 mm) and 12.8 +/- 2.8 mm (range 8.1-19.2 mm), respectively. Although the positive correlation between maximal jaw opening and facial morphology was significant, none of the variables significantly differed between the value of the condylar path and facial morphology. The length of the path of maximum incisor movement and the condylar path during mandibular movement also did not correlate. Stepwise multiple regression analysis indicated a positive association between the maximal length of the incisor path and the cephalometric value of mandibular ramus inclination (R2 value was 0.369). The results of this study suggest that facial morphology size has a limited effect on maximal voluntary mandibular opening and condylar movements in normal adult female subjects.

Polychlorinated biphenyls promote 1-nitropyrene-induced lung tumorigenesis without the induction of K-ras gene mutation in A/J mice.

Although the effects of polychlorinated biphenyls (PCBs) on human lung carcinogenesis are suggested from the massive PCBs poisoning that occurred in Japan designated "Yusho," the detailed molecular mechanism are unknown. 1 nitropyrene (1-NP), an ubiquitous and abundant environmental pollutant, is known to be detected in lung tissues derived from patients with lung cancer in Japan, and its relation to lung carcinogenesis is also suggested. We investigated the effects of PCBs (Kanechlor-400) on 1-NP-induced lung tumorigenesis in A/J mice. PCBs were administered intraperitoneally followed by ip injection of 1-NP. The lung lesions were examined 18 weeks after the final treatment. In the control group, no neoplastic lesions were induced in the lung. In the PCB group, preneoplastic lesions such as hyperplasia and adenoma were induced in 2/10 (20%) mice. In 1-NP group and in PCB + 1-NP group, lung lesions including adenocarcinoma were induced in 16/20 (80%) and 13/13 (100%) mice, respectively. Both the number and the size of tumors in PCB + 1-NP group were significantly greater than those in 1-NP group. K-ras gene mutation, CAA to CGA in codon 61 or GGT to GAT in codon 12, was found in either 1-NP group or PCB + 1-NP group but not in the PCB group. There was no difference in the pattern of K-ras mutation associated with the pretreatment with PCBs. These results suggest that PCBs promote 1-NP-induced lung tumorigenesis and may support, at least in part, the mechanism of the high incidence of lung cancer in patients with Yusho.

Micronucleus induction and chromosomal aberration of 1- and 3-nitroazabenzo[a]pyrene and their N-oxides.

Nitro-azabenzo[a]pyrenes, 1- or 3-nitro-azabenzo[a]pyrene and their N-oxides are nitrated derivatives of azabenzo[a] pyrene (ABP) containing nitrogen in the 6-position of benzo[a]pyrene (B[a]P). The nitro-ABP-N-oxides (ABPOs) were formed by reaction of ABP with excess HNO(3). These derivatives were noteworthy as potent mutagens for Salmonella strains, and were present in fine particles of diesel particulates. In this study, micronucleus induction in mice and chromosomal aberrations due to means of Chinese hamster lung fibroblast (CHL) cells were investigated to determine genotoxicity in order to define the relationship with the mutagenic potency of these derivatives. The induction of micronucleus polychromatic erythrocytes (MNPCEs) was dependent on the dose response of 10-40 mg for 3-N-6-ABP, and of 10-40 mg for 1-N-6-ABP, and in addition, 1- and 3-N-6-ABPOs markedly induced MNPCEs in a dose range of 10-400 mg and from 1 to 80 mg, respectively, when the compound was intraperitoneally administrated in two mice at each dose. The results show that of the four compounds, 3-N-6-ABPO demonstrated a marked increase in MNPCES: On the other hand, chromosomal aberrations of the four compounds were investigated by the duplicate tests using CHLS: The results after a 48 h treatment induced aberrations of the chromatid type, chromatid breaks and exchanges for 1- and 3-N-6-ABP, and mainly chromatid exchanges for 1- and 3-N-6-ABPO. The frequency of chromosomal aberrations associated with nitro substitution on the ABPO structure. Chromosomal aberrations of nitro derivatives of ABPO substituted at the 3-position on the structure were more potent than those at the 1-postion. N-oxide derivatives have been found to be reduced to anion radicals much more easily than azaB[a]P and its nitro derivatives. This suggests that the electrochemical reduction of the chemicals plays an important role in the metabolic activation of nitrated B[a]P derivatives.

[Effect of PCBs on mouse lung tumorigenesis induced by 1-nitropyrene: a preliminary report].

We have analyzed the effect of polychlorinated biphenyls (PCB, Kanechlor-400) on 1-nitropyrene (1-NP) induced lung tumor. Male A/J mice (6 weeks old) were used for the experiment. A total of 2.5 mg/kg PCB was administered intraperitoneally (PCB group), a total of 0.38 mmol/kg 1-NP was administered intraperitoneally for 17 times (1-NP group), PCB was administered followed by i.p. injection of 1-NP (PCB + 1-NP group), and only vehicle was administered (control group). The lung lesions induced were examined 18 weeks after the final treatment with 1-NP or vehicle. In control group, no neoplastic lesion in the lung was induced. In PCB group, only one lesion with adenoma was induced. In 1-NP group, various kinds of lung neoplastic lesions including hyperplasia, adenoma and adenocarcinoma were induced. In PCB + 1-NP group, both the number and size of tumors induced were significantly more than those in 1-NP group. In addition, the number of adenocarcinoma formed was more in PCB + 1-NP group than in 1-NP group. Each lesion was microdissected to collect and analyze DNA of the targeted tissue. K-ras gene mutation was detected in part of adenoma lesions and all the carcinoma lesions. The mutation was found in either 1-NP or PCB + 1-NP group, but not in control and PCB group. The pattern of K-ras mutation was CAA to CGA in codon 61 or GGT to GAT in codon 12. There was no difference in the pattern of K-ras mutation despite of the pretreatment with PCB. Although the present data are from small sample size, it was suggested that PCB may promote (but not initiate) 1-NP induced lung tumorigenesis, and may not induce K-ras mutation directly in the experimental system.

8-Hydroxyguanosine formed in human lung tissues and the association with diesel exhaust particles.

Diesel exhaust particles consist of various organic chemicals, heavy metals, and carbon particles. Knowledge of the fate of organic chemicals and carbon particles in the lungs is important to determine the mechanisms responsible for lung tumors. In the present study, diesel particle extracts were found to show mutagenicity for YG3003, a sensitive strain to some oxidative mutagens, as well as other mutant strains, and those of lung tissues obtained from lung cancer patients exhibited potent mutagenicity. Formation of 8-hydroxyguanosine (8-OHdG) as a biomarker of oxidative damage was analyzed with in vitro and in vivo assay systems. The 8-OHdG was detected in all 22 cases of lung tissues with carcinomas tested and their levels increased with the increasing age of the patients, suggesting a correlation between age and the presence of carbon particles in lung tissues. Therefore, the formation of 8-OHdG due to diesel exhaust particles was investigated via intratracheal injections into mice. 8-OHdG formation was elevated when carboneceous particles, after removal of organic chemicals with various solvents, were administered to mice, but it was not elevated when polyaromatic compounds such as benzo[a]pyrene, 1,8-dinitropyrene, and 1-nitropyrene were used in the same procedure in mice. The carboneceous particles were formed from a giant particle that was aggregated by micro-particles with diameters of 1.47 +/- 1.34 to 1.05 +/- 0.83 microm. These results suggest that carboneceous particles, but not mutagens and carcinogens, promote the formation of 8-OHdG, and that as a mechanism, alveolar macrophages may be involved in oxidative damage. The oxidative damage may be due to the fact that the mutation is involved with the generation of a hydroxyl radical during phagocytosis, and the hydroxyl radical leads to hydroxylation at the C-8 position of the deoxyguanosine residue in the DNA.

Analysis of environmental carcinogens associated with the incidence of lung cancer.

It is presumed that carcinogens present in human lungs contribute to the incidence of lung cancer. Most of the carcinogens are inhaled in lung alveoli with particulate matter through the respiratory tract. On the basis of chemical analysis of 256 lung specimens with carcinomas resected in the period 1991 1996, the concentration of 1-nitropyrene (1-NP) was 19.7+/-10.5 pg/g of dry weight, and that of the dinitropyrenes (DNP) was 3.50+/-0.14-6.26+/-1.76. In addition, 2-nitrofluoranthene (NF) and 3-NF were detected at a higher level of 38.6+/-17.2 and 39.1+/-14.2, respectively, pg/g of dry weight. Concentrations of benzo[a]pyrene, benzo[e]pyrene, benzo[k]fluoranthene and benzo[ghi]perylene in 37 specimens collected in the period 1991 1996, were in the range of 138+/-82-399+/-220 pg/g of dry weight. No difference was found in the concentration of chemicals deposited in lung specimens from patients with lung cancer and tuberculosis as a control. By following the prognosis of 112 patients with carcinomas, we found that the deposition of 1-NP, 1,3-DNP, and 3-NF in lung tissues influenced their 5-year-survival after determination of chemicals. Lung specimens were divided into two groups of higher and lower chemical concentrations at the levels of 18 pg/g for 1-NP, 15 for 1,3-DNP, and 35 for 3-NF, and the findings were statistically analyzed by adjusting for age, gender, smoking status and cell type. The 5-year-survival of patients was markedly lower in the higher concentration group than the lower group.

Comparative tumorigenicity of 1- and 3-nitrobenzo[a]pyrenes, and 3,6- and 1,6-dinitrobenzo[a]pyrenes in F344/DuCrj rats.

Our earlier study revealed that 1- and 3-nitrobenzo[a]pyrene (NBP), 1,6- and 3,6-dinitrobenzo[a]pyrene (DNBP), nitrated derivatives of benzo[a]BP (BP), are present in the environment. These derivatives are potent mutagens for Salmonella tester strains and we have preliminarily reported them to be carcinogenic in F344/DuCrj rats. In this study, the tumorigenic action of 1- and 3-NBP, 1.6- and 3,6-DNBP, and BP induced by subcutaneous injection into rats was found to differ according to the NO2-substitution in the BP structure. The chemicals were suspended in equal volumes of beeswax and tricaprylin, and rats were subcutaneously injected with single doses of 500, 1000, and 2000 microg for 1- and 3-NBP, and of 8, 40, 200, and 1000 microg for 3,6- and 1,6-DNBP, and BP as a positive control. 3,6-DNBP and BP induced tumors in a dose-dependent manner at the injection site. Rats given 1000 microg of 3,6-DNBP (2924 nmol) and BP (3968 nmol) developed subcutaneous tumors at the rate of 70 and 80%, respectively, and those given a minimum dose of 23 nmol for 3.6-DNBP and 32 nmol for BP per rat developed tumors at a rate of 4.8 and 18.2%, respectively. However, rats given 500 and 1000 microg of 1- and 3-NBP did not develop any tumors while those given a high dose, 2000 microg, of each chemical developed tumors at only one of ten animals used. It was concluded, therefore, that these chemicals are weak carcinogens. Histologically, most of the tumors were malignant fibrous histiocytomas. Rats given various doses of 1,6-DNBP did not develop any tumors at the injection site. The failure of 1,6-DNBP to induce tumors may involve its metabolites because of the lower mutagenicity of its reduction products, 1-nitroso-6-NBP and 1-amino-6-NBP. It is suggested, therefore, that tumorigenicities of NBPs and DNBPs differ according to the NO2-substitution on the chemical structure, which may be due to the possible nitroreduction of the chemicals.

Ki-ras mutation and cell proliferation of lung lesions induced by 1-nitropyrene in A/J mice.

In this study, lung lesions were found in male A/J mice 24 wk after intraperitoneal injection of 1-nitropyrene (1-NP). The lesions were classified into three categories: alveolar/bronchiolar hyperplasia, adenoma, and adenocarcinoma. The proliferation kinetics of cells in the lesions were evaluated by assessing proliferating cell nuclear antigen (PCNA) expression and silver-staining nucleolar organizer regions (AgNORs). Furthermore, the role of the Ki-ras gene in tumorigenesis was studied by detecting point mutations in Ki-ras codons 12, 13, and 61 by polymerase chain reaction and sequence analysis. The PCNA-positive rates (+/- standard deviations) in various samples were as follows: 0% for specimens from six untreated animals and six uninvolved areas, 4.26 +/- 3.94% for 19 hyperplasias (hyperplasias vs normal lung tissue, P < 0.01), 13.24 +/- 6.35% for 25 adenomas (adenomas vs hyperplasias, P < 0.01), and 38.0 +/- 9.63% for four adenocarcinomas (adenocarcinomas vs adenomas, P < 0.01). The corresponding mean AgNOR scores were as follows: 1.10 +/- 0.05 for the untreated animals, 1.32 +/- 0.09 for the uninvolved areas, 1.72 +/- 0.59 for the hyperplasias (hyperplasias vs normal lung tissue, P > 0.05), 2.74 +/- 0.70 for the adenomas (adenomas vs hyperplasias, P < 0.01), and 5.22 +/- 0.62 for the adenocarcinomas (adenocarcinomas vs adenomas, P < 0.01). Ki-ras gene mutations were identified in three of four (75%) adenocarcinomas, six of 23 (26%) adenomas, and two of 17 (12%) hyperplasias. No mutations were found in normal lung tissue. The most frequent Ki-ras mutation was an arginine (CGA)AT --> GC transition at codon 61 in exon 2. The PCNA-positive rates and AgNOR scores of cases with Ki-ras mutations were higher than those without an identified mutation (P < 0.05). Ki-ras mutations at codon 61 (Arg) may therefore influence the growth or development of 1-NP-induced lung lesions in A/J mice.

Possible role of indoor environment and coal combustion emission in lung carcinogenesis in Fuyuan County, China.

Fuyuan Country, in Yunnan Province, China has an extremely high lung cancer mortality both in males and non-smoking females. Out of 5768 deaths, 588 patients died of malignant diseases. Lung cancer was the number one cause of death among malignant diseases both in males and females. The rate of lung cancer death to the whole of malignant diseases was 56.2% for males and 55.0% for females. Indoor soot and combustion emission derived from smoky coal produced in northern Fuyuan exhibited high mutagenic activities against Salmonella typhimurium TA98 strain in Ames test. Resected lung tissues derived from the patients with lung cancer in Fuyuan contained significantly higher concentrations of benzo(a)pyrene than those in Japan, both in males and females (i.e., 608.7 +/- 477.1 pg/dry weight for samples of the patients in Fuyuan, 180.1 +/- 104.5 for Japanese non-smokers, and 207.5 +/- 98.8 for Japanese heavy smokers, respectively). These results suggest that mutagenic chemicals contained in coal as well as indoor environment may have a great influence on lung carcinogenesis in Fuyuan, Yunnan Province, China.

Mutagenicity of the fullerene C60-generated singlet oxygen dependent formation of lipid peroxides.

Fullerene C60 dissolved in polyvinylpyrrolidone was mutagenic for Salmonella strains TA102, TA104 and YG3003 in the presence of rat liver microsomes when it was irradiated by visible light. The mutagenicity was elevated in strain YG3003, a repair enzyme-deficient mutant of TA102. The mutation was reduced in the presence of beta-carotene and parabromophenacyl bromide, a scavenger and an inhibitor, respectively, of phospholipase. The results suggest that singlet oxygen was generated by irradiating the C60 by visible light and that the mutagenicity was due to oxidized phospholipids in rat liver microsomes. Of the phospholipids in rat liver microsomes, the linoleate fraction isolated by high performance liquid chromatography was a major component, and played an important role in mutagenicity. Methyl linoleate, which was prepared for gas chromatographic analysis, was readily oxidized to hydroperoxymethyl linoleate, and associated with both 10- and 12-hydroxyl derivatives with a double bond in chemical structure by singlet oxygen: radicals to the hydroxyl function were probably generated. Because of the instability of the hydroxymethyl linoleate radicals, guanine residues generated radicals. The results of ESR spectrum analysis suggested generation of radicals at the guanine base but not thymine, cytosine and adenine bases as estimated with the g value of 2.0150. On the other hand, the singlet oxygen-generating C60 formed 8-hydroxydeoxyguanosine (8-OH-dG) upon treatment with 2' deoxyguanosine and microsomes or linoleate. The formation of 8-OH-dG was highly elevated in the presence of microsomes and linoleate. The level of 8-OH-dG formed with and without the microsome fraction was 47 and 9.6 units, respectively, per 10(4) deoxyguanosine. It was considered that the mechanism is indirect action of singlet oxygen due to lipid peroxidation of linoleate that causes oxidative DNA damage.

Mutagenicity of nitrophenanthrene derivatives for Salmonella typhimurium: effects of nitroreductase and acetyltransferase.

To determine the mutagenicity of nitrophenanthrenes, three mononitrophenanthrenes (NPhs), 11 dinitrophenanthrenes (diNPhs) and eight trinitrophenanthrenes (tiNPhs) were synthesized, and their mutagenicity was investigated by using Salmonella typhimurium his- strains TA98, TA100, and TA98NR, nitroreductase-deficient, and TA98/1,8-DNP6, O-acetyltransferase-deficient mutants, and strains YG1021 and YG1026, nitroreductase-overproducing mutants of TA98 and TA100, respectively, and strains YG1024 and YG1029, O-acetyltransferase-overproducing mutants of TA98 and TA100, respectively. 1-, 3- and 9-NPhis induced 329, 620 and 438 revertants per nmol in strain TA100, respectively, and 4,839, 11,309 and 16728 revertants per nmol, respectively, in strain YG1029. Mutagenicity of 1,6-, 2,6-, 2,9-, 2,10-, 3,5-, 3,6- and 3,10-diNPh was elevated in strains YG1021, YG1024, YG1026 and YG1029. Among these derivatives, 1,6-, 2,6-, 3,6- and 3,10-diNPhs were more mutagenic in strains YG1024 and YG1029 than YG1021 and YG1026, and they showed a structure-activity relationship between mutagenicity and NO2-substitution. Nitro derivatives substituted at the 3 and 6 positions of their chemical structure strongly mutated both strains YG1024 and YG1029, whereas those substituted at the 9 and 10 positions showed weak mutagenicity. In addition, nitro substituents at positions 4 and 5 were perpendicular while those on positions 2,3,6 and 7 were nearly coplanar to the aromatic ring. Furthermore, 2,6,9-, 3,6,9- and 1,6,9-trinitrophenanthrenes (triNPhs) were mutagenic for strain TA100, and their mutagenicity was more enhanced in YG1024 and YG1029 than in YG1021 and YG1026. Of the eight triNPhs all except 1,5,10-triNP were mutagenic in TA98 and TA100, and their mutagenicity was more enhanced in YG1024 and YG1029 than in YG1021 and YG1026. These results suggest that these compounds are mutagens that are activated by O-acetyltransferase esterification following nitroreductase. The nitrated derivatives substituted at the 2(7) and 3(6) positions of the phenanthrene ring were highly mutagenic. The relationship between chemical structure and the mutagenicity of NPh derivatives is discussed.

Moderate inhibition of mutagenicity and carcinogenicity of benzo[a]pyrene, 1,6-dinitropyrene and 3,9-dinitrofluoranthene by Chinese medicinal herbs.

The activity of six Chinese medicinal herbs against the environmental mutagens and carcinogens benzo[a]pyrene (B[a]P), 1,6-dinitropyrene (1,6-diNP) and 3,9-dinitrofluoranthene (3,9-diNF) was determined. Samples of Prunella spica, Rheum palmatum, Polygonum multiflorum, Agrimonia pilosa, Ephedra sinica and Teitoutou were tested in an in vitro system. Antimutagenic activity against B[a]P was marked in the presence of extracts (boiled for 2 h in a water bath) whereas that against 1,6-diNP and 3,9-diNF varied from 20 to 86%. The differences in inhibition might be due to inactivation of metabolic enzymes. An extract of P. multiflorum was divided into ether, ethyl acetate and water soluble fractions, which were tested for antimutagenic activity against B[a]P. The antimutagenic action of the ethyl acetate soluble fraction was substantial and dose-dependent. Tannins and related compounds were the major components of the extract, of which epigallocatechin, epigallocatechin gallate, epicatechin gallate and tannic acid strongly inhibited the mutagenicity of B[a]P (2.5 micrograms/plate) in Salmonella typhimurium TA98 with S9 mix. To confirm the results of the in vitro test system, F344/DuCrj male rats were given a subcutaneous injection of B[a]P. Thereafter, they received water extracts of the six Chinese medicinal herbs for 50 weeks and were examined for tumors. The P. multiflorum extract significantly reduced the tumor incidence.

Mutagenic and carcinogenic significance and the possible induction of lung cancer by nitro aromatic hydrocarbons in particulate pollutants.

Studies of genotoxicity and carcinogenicity of nitro aromatic hydrocarbons focus on their high mutagenicity for bacteria and mammalian cells. Nitrobenzo[a]pyrenes (NBPs) and related nitroazaarenes also are extraordinarily mutagenic. 3-Nitro-6-azabenzo[a]pyren-N-oxide was found to be a more potent mutagen than 1,8-dinitropyrene. Mutagenicity of NBPs was associated with the position of substitution of the nitro function when nitrogen dioxide (NO2) was substituted at the third position on the benzo[a]pyrene (BP) structure, as in 3,6-dinitrobenzo[a]pyrene but not in 1,6-diNBP. The NBPs were reduced by a rat liver postmitochondrial fraction to nitroso- and subsequently to amino-derivatives. Therefore, tumoral action in rats was induced at significant levels by subcutaneous injection of 3,6-diNBP, but no tumors were observed in rats given 1,6-diNBP. Carcinogenic nitropyrenes were detected in the resected lung of a patient with lung cancer. It is suggested that the presence of nitropyrenes and the resulting tumor were due to exposure to by-products of combustion of heavy oil. The patient was a nonsmoker and farmer who had bred chickens for 40 years. He used heavy oil for heating the chicken house. Similarly, a group of Chinese people at high risk of developing lung cancer was selected to determine the initiator of lung cancer. Lung cancers were obtained from six Chinese female nonsmokers who were living in Fuyuan County, China. Polycyclic aromatic hydrocarbons were detected in resected lung specimens; they were benzo[k]fluoranthene, BP, benzo[g,h,i]perylene, and pyrene. These cases were associated with exposure to soot from combustion of coal usually used for heating and cooking indoors.

Detection of nitro-azabenzo[a]pyrene derivatives in the semivolatile phase originating from airborne particulate matter, diesel and gasoline vehicles.

Mutagens in the semivolatile phase of airborne particulate matter, diesel and gasoline engine emissions were investigated using chemical and biological assays. The previously unknown mutagens, 1-(3-)nitro-6-azabenzo[a]pyrenes (1-N-6-ABP and 3-N-6-ABP) and 1-(3-)nitro-6-azabenzo[a]pyrene-N-oxides (1-N-6-ABPO and 3-N-6-ABPO), were detected in the semivolatile phase, which was adsorbed onto XAD-4 resin combined with a Teflon-coated fibre filter. Dichloromethane extracts of materials adsorbed onto XAD-4 resin were divided into acidic, neutral and basic by liquid-liquid separation. These chemicals in the basic fraction were the major mutagens in the semivolatile phase when they were bioassayed using Salmonella typhimurium TA98 in the absence of S9 mix; the mutagenicity of the basic fraction contributed at the rate of 42.9-68.8% of that of crude extracts for airborne, diesel and gasoline emission materials. As the mutagens were found to be difficult to analyse by gas chromatography (because of their probable absorption onto the packing) they were purified by HPLC and analysed by mass spectrometry. The concentrations of 1-N-6-ABP, 3-N-6-ABP, 1-N-6-ABPO and 3-N-6-ABPO detected were 1.1, 1.2, 0.8 and 0.3 ng/g, respectively, of materials in the airborne sample, and 4.9, 7.7, 2.2 and 3.8 ng/g, respectively, of materials in the diesel emission. Only 1-N-6-ABP (3.4 ng/g) and 3-N-6-ABP (4.9 ng/g) were detected in the semivolatile phase of the gasoline engine emission.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of mutagens/carcinogens in the excised lung and analysis of lung cancer induction.

To elucidate a relationship between lung cancer and tumor induction of environmental chemicals, the presence of 1-nitropyrene (1-NP) and benzo[a]pyrene (B[a]P) in 137 Japanese (97 male and 40 female), and 21 Chinese lung specimens was examined by HPLC, and GC-MS and environmental exposure discussed. Mortality due to lung cancer in Fuyuan County, China, is much higher than that of other cancers. We investigated 21 patients who were residents of Fuyuan County. All were female aged 28-64 years and were non-smoking farmers and cooks. The histological features of the tumors were adenocarcinoma, squamous cell carcinoma and small cell carcinoma. The incidence of cancer was due to inhaling soot from the combustion of coal used for cooking and indoor heating. The polycyclic aromatic hydrocarbons, chrysene, benzo[k]fluoranthene, B[a]P, benzo[g,h,i]perylene and pyrene, but not their nitrated derivatives, were detected in substantial amounts in the resected lungs. These mutagens and carcinogens normally originate in combustion products of coal, and are discussed as the possible initiators of the tumors in the lungs of these patients. All the Japanese lung specimens were also obtained from non-smokers. The concentrations of 1-NP averaged 21.3 +/- 12.4 and 5.9 +/- 2.4 pg/g of Japanese and Chinese samples respectively. The concentrations of B[a]P averaged 180.2 +/- 103.7 and 608.7 +/- 477.1 pg/g of Japanese and Chinese samples respectively. Thus, Japanese and Chinese lung specimens were mainly contaminated by 1-NP and B[a]P, respectively. Typical tissues from a carcinomatous human lung were examined. The patient was a 64-year-old Japanese male non-smoker and farmer who had raised chickens over a period of 40 years. The histological features of the tumor were those of keratinizing squamous cell carcinoma. 1-Nitropyrene, 1-nitro-3-hydroxypyrene, 1,3-dinitropyrene and chrysene were detected at concentrations of 0.11, 0.036, 0.095 and 0.16 ng respectively per gram of lung tissue. This cancer was due to long-term exposure to the combustion products of heavy oil used in a chicken house.

[Therapeutic trials for promotion of faecal excretion of PCDFs by the administration of rice bran fiber and cholestyramine in Yusho patients].

It is well-known that Yusho disease was caused by polychlorinated dibenzofurans (PCDFs), and that 2, 3, 4, 7, 8-Pentachlorodibenzofuran (PnCDF), 1, 2, 3, 4, 7, 8- and 1, 2, 3, 6, 7, 8-Hexachlorodibenzofurans (HxCDFs) still retain in the patient bodies. As patients usually suffer from various chronic syndrome, an effective treatment is extremely needed. In order to assess the rice bran fiber (RBF) and cholestyramine on stimulating faecal excretion of PCDFs, two clinical trials were carried out in 1990 and 1991. In the first trial in 1990, 10 g of RBF (dietary fiber content was 50%) and 4 g of cholestyramine were administered to four Yusho patients three times a day for a week. The stool from patients were collected a week before and during the administration. These were pooled respectively, and then two samples for measurement. In the second trial in 1991, 10 g of dietary fiber rich RBF (refined-RBF, dietary fiber content was 85%) and 4 g of cholestyramine were administered to four Yusho patients three times a day for two weeks. In this trial, three stool samples were obtained from each patient, ie., a week before administration, and first and second week during administration. Level of PCDFs was determined by high resorption GC/MS and the following results were obtained. 1) In the first trial (1990) the faecal excretion of PnCDF and HxCDFs increased at the rates of 42-88% and 7-47%, respectively, in three out of four patients.(ABSTRACT TRUNCATED AT 250 WORDS)

[Stimulating effect of dietary fiber on fecal excretion of polychlorinated dibenzofurans (PCDF) and polychlorinated dibenzo-p-dioxins (PCDD) in rats].

We investigated the stimulating effect of dietary fiber on fecal excretion of PCDF and PCDD stored in the rat body. Twenty-eight male rats (71-74g body weight) were orally administered with 1ml of the causal rice oil of Yusho desease. The rice oil was contaminated with 2, 3, 4, 7, 8-pentaCDF (691.4ng), 1, 2, 3, 4, 7, 8-hexaCDF (708.6ng), 1, 2, 3, 6, 7, 8-hexaCDF (128.4ng), 1, 2, 3, 7, 8-pentaCDD (7.2ng), 1, 2, 3, 6, 7, 8-hexaCDD (34.1ng), 1, 2, 3, 7, 8, 9-hexaCDD (20.1ng) and 1, 2, 3, 4, 6, 7, 8-heptaCDD (115.9ng). The animals were fed a control diet containing 10% cellulose for seven days. Twenty-eight rats consisting of four rats a group were housed and rats of each group were given a treatment diet containing 10% rice-bran-fiber (RBF), 5% cholestyramine, 10% RBF + 5% cholestyramine, 10% RBF + 5% cholestyramine + 1% squalane, 10% burdock-fiber, 10% corn-fiber and 10% soybean-fiber during a period from eight to twenty-one days. The remaining four rats served as controls. PCDF and PCDD in feces, liver, small intestine and gastrointestinal tract were analyzed by high resolution gas chromatography-mass spectrometry. PCDF level in small intestine of rats administered with RBF + cholestyramine showed a decrease of 40% over the level of control rats.(ABSTRACT TRUNCATED AT 250 WORDS)