Analysis of automobile exhaust condensates.

1. On the basis of figures for the production of PAH during the Europa drive cycle by 100 passenger cars and those for the consumption of petrol in the Federal Republic of Germany, an annual emission of 1,850 kg benzo[a]pyrene from petrol engine vehicles has been calculated. 2. The carcinogenic effect of benzo[a]pyrene accounts for only 9% of the total activity of exhaust condensates. 3. The amounts of other known carcinogenic PAH, such as benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[j]fluoranthene, indeno[1,2,3-cd]pyrene and dibenzo[a,h]anthracene are shown in Table 2. (table: see text). Assuming that there is no significant promoting or hyper-additive effect, it can be estimated that these six known carcinogenic PAH contribute about 10-15% of the total carcinogenicity. 4. Six unknown PAH were found: cyclopenteno[cd]pyrene (mol wt 226), methylenebenzo[a]pyrene (mol 264), methylenebenzo[e]pyrene (mol wt 264), methylenebenzo[ghi]perylene (mol wt 288), PAH mol wt 300A and PAH mol wt 300B. It is reasonable to assume that these unknown PAH account for the predominant part of the carcinogenic effect. Biological tests with these pure substances are being undertaken by Drs Pott, Pfeiffer and Habs. 5. It has been shown that almost all of the carcinogenic effect of automobile exhaust condensates is due to PAH. To support this claim, the carcinogenic effects of the exhaust condensate should be compared with those of a mixture of the known and unknwon PAH in the same proportions as are found in the exhaust condensate. The gas chromatogram of such a mixture is shown in Figure 6.     

Tumours in mice after subcutaneous injection of automobile exhaust condensates.

Automobile exhaust condensate (AEC), either mixed with benzo[a]pyrene (BaP) or suspended or dissolved in tricaprylin, was injected subcutaneously into NMRI mice in a series of experiments. The addition of AEC decreased the incidence of tumours which developed with 30, 90 and 270 microgram BaP. Reduction of tumour incidence was proportional to the amount of AEC added. With an injection of 10 microgram BaP, the latent period was greatly increased when AEC was added, but the occurrence of tumours was the same. Components of AEC appear to inactivate BaP, at least temporarily. In further experiments AEC and nine fractions thereof were injected subcutaneously into mice. The fraction comprising only polycyclic aromatic hydrocarbons (PAH) induced the highest incidence of tumours. In contrast, when it was administered in combination with other fractions the PAH fraction was less active. Application of the products of further fractionation of PAH showed that polycyclic compounds with seven or more rings can also induce tumours in this model.     

Syncarcinogenic action of polycyclic hydrocarbons in automobile exhaust gas condensates.

The carcinogenic action of the hydrocarbon mixture predominantly found in condensates of automobile exhaust gas can be attributed almost exclusively, on the basis of the results presented here, to a group of substances (PAH) with an insignificant weight proportion. It was not possible to demonstrate an inhibitory action of most of the weak-to-inactive hydrocarbons; on the contrary, an additive effect of the two types could be observed. At very high doses (almost 10 times higher than the highest doses selected in the rest of the trial) the group of substances which were supposed to be non-carcinogenic also proved to be biologically effective. Subsequent individual studies on the same animal strain should clarify whether this action is to be attributed to one or two representatives of this group, e.g., benzo[e]pyrene or chrysene.     

Direct-acting mutagens in automobile exhaust.

Particulate matter in city air contains chemicals which are mutagenic in the Ames Salmonella typhimurium assay. In residential urban areas, the principal mutagens in air do not require liver enzymes to be activated. The source of these liver independent (direct-acting) mutagens may be automobile exhaust because (1) the mutagenic activities were correlated to the lead content or air, (2) the mutagens were found exhaust samples from automobiles and from an experimental CFR single-cylinder gasoline engine, and (3) these mutagens were not found in fuel or unused motor oil, but were found in used motor oil. The strain specificity and the fact that liver enzymes were not required for activation indicated that the exhaust and airborne mutagens were not unsubstituted polycyclic aromatic hydrocarbons (PAH), aromatic amines, alkylnitrosamines or aliphatic epoxides, peroxides and hydroepoxides. A number of nitro-substituted aromatic compounds are direct-acting mutagens in the Ames test, and it is possible that nitration of PAH in exhaust may form the compounds observed here. We synthesized 6-nitrobenzo [a] pyrene and found it to be a potent, direct-acting mutagen with activity comparable to that of benzo-[a] pyrene.     

Pulmonary tumorigenesis in Syrian golden hamsters after intratracheal instillations with automobile exhaust condensate.

Syrian golden hamsters were intratracheally instilled once every two weeks for life with automobile exhaust condensate (AEC) at two dose levels. The condensate was prepared from the most common German passenger car driven in the Europa-Test cycle (simulated city driving) and contained 340 microgram/g benzo(a)pyrene (B(a)P). Despite the very low total dose of B(a)P received, all animals of both dosage groups developed multiple pulmonary adenomas. Electron microscopy revealed these tumors to resemble B(a)P induced pulmonary adenomas in their ultrastructure. Numerous condensate-laden macrophages in tumor and peripheral lung tissues indicated that little of the instilled condensate had been removed from the airways through mucociliary action.     

32P-postlabelling analysis of DNA adducts in the skin of mice treated with petrol and diesel engine lubricating oils and exhaust condensates

Samples of unused or used petrol and diesel engine lubricating oils were applied to the shaved dorsal skin of 4- to 6-week-old male Parkes mice, either as a single treatment (50 microliters/mouse) or as four consecutive daily treatments (50 microliters/application). DNA isolated from the skin 24 h after the final treatment was digested to 3'-mononucleotides and analysed by 32P-postlabelling for the presence of aromatic adducts. Enhancement of sensitivity using butanol extraction or nuclease P1 digestion of the DNA hydrolysates led to the detection of up to eight adduct spots on polyethyleneimine-cellulose thin-layer chromatograms with samples of DNA from skin treated with used engine oils, at levels of 40-150 amol total adducts/micrograms DNA. Multiple treatments with the used oils gave rise to similar patterns of adducts in lung DNA. A single treatment of mouse skin with petrol engine exhaust condensate (50 microliters), or diesel engine exhaust condensate (50 microliters), containing 20 and 46 micrograms benzo[a]pyrene (BaP)/g respectively, gave rise to approximately 75 amol total adducts/micrograms DNA in skin. A significant proportion, 31 and 48% respectively, of the adducts formed by the petrol and diesel engine exhaust condensates co-chromatographed with the major BaP-DNA adduct, but with the used engine oils, only petrol engine oil, and not diesel engine oil, produced significant amounts of an adduct (22% of total) that corresponded to the BaP-DNA adduct.     

Lung carcinogenesis and formation of 8-hydroxy-deoxyguanosine in mice by diesel exhaust particles

In order to clarify the involvement of oxygen radicals in lung carcinogenesis induced by diesel exhaust particles (DEP), the relationship between lung tumour response and formation of 8- hydroxydeoxyguanosine (8-OHdG) in lung DNA was examined. The role of high dietary fat and beta-carotene on these responses was also studied. Mice were intratracheally injected with 0.05, 0.1 and 0.2 mg of DEP per animal once weekly for 10 weeks. After 12 months, the lung tumour incidence in mice treated with 0.05 mg and 0.1 mg showed similar increases (30% and 31%), but was decreased to 24% at 0.2 mg. High dietary fat enhanced the incidence of both benign and malignant tumours. beta-carotene partially prevented the tumour development. After the 10 weekly treatments of DEP, inflammatory reaction was observed in the respiratory tract and alveoli. The formation of 8-OHdG in lung DNA from mice treated with DEP showed a dose dependent increase. 8-OHdG formation was enhanced by high dietary fat and partially reduced by beta-carotene. Formation of 8-OHdG was significantly correlated with the lung tumour incidence except at 0.2 mg. These results suggest that the induction of oxidative DNA damage may be an important factor in the initiation of DEP-induced lung carcinogenesis, and that beta-carotene and high dietary fat may play a role in the regulation of tumour development via modulation of the formation of 8-OHdG.      

Ultrasound-guided percutaneous microwave ablation of adrenal metastasis: Preliminary results

Purpose: To evaluate the feasibility, safety and therapeutic effects of ultrasound (US)-guided percutaneous microwave (MW) ablation in the treatment of adrenal metastasis.

Materials and methods: From May 2006 to April 2008, five consecutive patients with pathologically proven unilateral adrenal metastases with a diameter of 2.3 to 4.5 cm were treated by US-guided percutaneous MW ablation. Four metastases were in the right side, one metastasis was in the left side. For each application, two cooled-shaft needle antennae were percutaneously inserted into the tumour under real-time US guidance. One thermocouple needle was inserted at the periphery of the tumour to monitor temperature in real-time during MW ablation. MW emission was ended when the entire tumour became hyperechoic and the temperature at the tumour border reached 54°C for at least 3 min. Technical success was defined as loss of tumour enhancement on contrast-enhanced imagings.

Results: All adrenal metastases were completely ablated after scheduled MW ablation sessions (mean, 1.2 sessions, range, 1 to 2 sessions). No major complications related to MW ablation occurred. In a median follow-up of 19 months (range 8 to 31 months), persistent absence of tumour enhancement was observed in the treated tumour in all patients.

Conclusions: US-guided percutaneous MW ablation appears to be a safe and effective therapy in selected adrenal metastasis.     

Hepatocellular carcinoma results from chronic cyclin D1 overexpression in transgenic mice.

Cyclin D1 is a known oncogene and a key regulator of cell cycle progression. Amplification of the cyclin D1 gene and its overexpression have been associated with aggressive forms of human hepatocellular carcinoma (HCC). In this study, two independent lines of transgenic mice have been generated that express cyclin D1 under the control of the rat liver fatty acid binding protein promoter. This transgene specifically directs expression in the liver and the intestines. RNA and protein analysis demonstrated increased expression of the cyclin D1 gene product in the liver and bowel when compared with wild-type siblings. Both transgenic lines developed progressive liver disease. Examination of H&E stained sections of the liver and bowel revealed hyperplastic changes in the liver by 3 months of age. By 6 months of age, transgenic mice had obvious hepatomegaly and histological evidence of dysplasia in the liver. These early changes were significantly more dramatic in male animals when compared with female animals. By 9 months of age adenomas of the liver appeared, progressing to HCC over the ensuing 6-month period. By 15-17 months of age, 87% of male and 69% of female animals had either adenomatous nodules or HCCs. By 17 months of age, 31% of male and female animals had disease that had progressed to HCC. These animals represent a unique and significant new model for the study of human HCC. This study demonstrates that overexpression of cyclin D1 is sufficient to initiate hepatocellular carcinogenesis.     

Transperineal percutaneous iridium-192 interstitial template implant of the prostate: results and complications in 321 patients.

A total of 321 patients with localized adenocarcinoma of the prostate treated by modified pelvic lymphadenectomy, Iridium-192 implant, and external beam iridium radiation were retrospectively reviewed. Analysis covered 8 years between 1981 and 1989 with a median population age of 72 (range 42 through 82 years). Disease-free survival for the entire group is 69% at 5 years with a median follow-up of 34 months (range 1.5 months to 98.5 months). As expected, both bulkier disease and positive nodal status adversely affected 5-year disease-free survival (p = 0.0001 for both). For tumors stage T1b (A2), T2a (B1), T2b (B2), T3 (C) the disease-free survival is 89.5%, 89.9%, 64.7%, and 48.8%, respectively; for NO disease 5-year disease-free survival is 76.5% versus N1/N2 disease with 5-year disease-free survival of 33.2%. Local control was excellent except for bulkier disease (p = 0.009). Tumors T1b, T2a, T2b, and T3 have 60-month local control rates of 95%, 93%, 83.6%, and 73.1%, respectively. Histologic grade also affected disease-free survival and local control with grade 1, grade 2, grade 3 showing 81.2%, 65.7%, and 45.1% disease-free survival at 5 years; and 93.6%, 82.2%, and 72.4% local control at 5 years. Estimates obtained using Kaplan-Meier method. Radiation induced morbidity was analyzed separately for all patients, there were 41 patients (13% of total) with 54 documented complications. There were no Grade 4 or 5 complications as per RTOG categories. Only 3 cases showed grade 3 complications (1%) and 51 cases showed grade 2 complications (15.9%). Grade 1 complications were not recorded. Of the grade 2 and grade 3 complications 30 were GU and 22 were rectal. The morbidity associated with combined interstitial implantation by transperineal percutaneous template and external beam iridium radiation for the localized prostate cancer is minimal with excellent local control and disease-free survival.     

Inhibition of intercellular communication in human keratinocytes by fractionated asphalt fume condensates.

Asphalt fume condensate (AFC) and chromatographically separated fractions have been shown to cause cancer in mouse skin. The levels of known carcinogenic initiators in these complex mixtures, however, are considered too low to account for their carcinogenic potency. It has been proposed that AFC may contain co-carcinogenic or tumor-promoting agents in addition to carcinogenic initiators. Modulation of gap junctional intercellular communication (GJIC) has been implicated as an important effect of tumor promoters. In this study, we examined the effect of five chromatographically generated fractions of AFC on GJIC in cultured human epidermal keratinocytes (HEK). HEK cells were exposed overnight to medium containing DMSO extracts of AFC fractions. GJIC was evaluated by dye-coupling of microinjected Lucifer Yellow CH. All AFC fractions produced a concentration-dependent inhibition of GJIC. The apparent potency of each fraction correlated with its relative polarity based on HPLC elution characteristics. Cells with reduced GJIC as a result of AFC fraction exposure were found to exclude propidium iodide, suggesting that inhibition of GJIC occurred in the absence of cell killing. However, significantly reduced culture DNA content was found following the overnight exposure to the highest concentrations of AFC fractions C, D and E.     

Combined 5-fluorouracil/systemic interferon-beta gene therapy results in long-term survival in mice with established colorectal liver metastases.

Preclinical in vitro and in vivo studies have demonstrated synergistic interactions between 5-fluorouracil (5-FU) and type I and II IFNs against human colorectal cancer cells. Despite these activities, randomized human trials have failed to identify a clinical benefit for this combination treatment. These limited clinical results may be secondary to the short half-life of recombinant IFN protein and the increased systemic toxicities of 5-FU/IFN combinations. We have previously reported an adenoviral-mediated IFN-beta gene therapy strategy, which may circumvent the pitfalls of recombinant IFN therapy. However, a dose-dependent toxicity and acute inflammatory response to systemically administered adenovirus vectors may limit the clinical application of this therapy. The combination of adenoviral-mediated IFN-beta gene therapy and 5-FU resulted in tumor regression, apoptosis, and improved survival in an established liver metastases model. These therapeutic effects were observed at a significantly lower vector dose than we had previously reported and with limited toxicity. This approach may allow for an effective clinical application of this therapy and warrants additional investigation.     

On the contribution of polycyclic aromatic hydrocarbons to the carcinogenic impact of automobile exhaust condensate evaluated by local application onto mouse skin

The objective of this investigation was to identify the substances chiefly responsible for the carcinogenicity of automobile exhaust condensate using topical application onto the skin of mice. This was performed by comparing the carcinogenic effect of various fractions with that of an unseparated sample of automobile exhaust condensate, tested in 3 different doses. The probit and Weibull analysis of the result shows: (a) The condensate, emitted from a gasoline-driven automobile provokes local tumors after long-term application to the dorsal skin of mice. The tumor incidence demonstrates a clear cut dose-response relationship. (b) The fraction of polycyclic aromatic hydrocarbons (PAH) containing more than 3 rings accounts for about 84-91% of the total carcinogenicity of automobile exhaust condensate. This fraction represents only about 3.5% by wt of the condensate. (c) The content of benzo[a]pyrene (BaP) (0.414 mg/g) accounts for 6-7.6% of the total carcinogenicity of automobile exhaust condensate, 15 selected PAHs for about 41%. (d) Regarding the minor effect of the PAH-free fraction (about 83% by wt), no hints for a cocarcinogenic activity were observed.