Mutagenic Index Research Articles

Toxicological and mutagenic analysis of Artemisia dracunculus (tarragon) extract

Mutagenicity and liver toxicity of the herb tarragon (Artemisia dracunculus) were evaluated using single cell gel (comet) electrophoresis. Ten microlitres aliquots of peripheral venous human blood were incubated with tarragon extract, saline, or the mutagen sodium dichromate. Cell suspensions dispersed in low-melting agarose were electrophoresed in ethidium bromide. The resulting DNA migration trails were obtained using fluorescent microscopy at 400× magnification, and graded according to the mutagenicity index (MI) for each cell incubation condition. The in vivo liver toxicity of Artemisia dracunculus was assessed in the blood of mice treated orally with the extract of the herb, using alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as liver function indicators. Liver morphology was assessed using hematoxylin and eosin (HE) staining of liver tissue. The present study demonstrated a direct correlation between tarragon extract dosage and three major outcome variables: MI; serum liver enzyme activity; and liver histopathology. These outcomes are possibly due to the presence in tarragon of methylchavicol and other genotoxic compounds. These findings provide a preliminary guide for risk assessment of tarragon in diet and in possible therapeutic applications.

Effect of Temperature, Fuming Time, and Distance from Source on the Polycyclic Aromatic Compound (PAC) Content of Bitumen Fumes: Assessment Using a Laboratory Fuming Protocol in Combination with a Sensitive Method for Determining Relative Aggregate Levels of PACs in Complex Mixtures

A laboratory fuming procedure was used in combination with a previously reported variation of the Ames Test (the Nitration Assay) to investigate the relationship between temperature, time at temperature, and distance from source, on the relative PAC content of fumes generated from paving bitumens. The test method, whose endpoint is mutagenic potency (Nitration Mutagenicity Index, NMI), takes advantage of large amplifications of Ames Test mutagenicity resulting from nitro-derivatization of PACs. NMIs were similar and quite low for fumes generated at 110°C, but increased exponentially up to 160°C. NMIs increased linearly with fuming time up to 40 min, after which they remained constant. With some initial fluctuation, they also remained constant with increasing distance of the condenser from the bitumen surface up to 100 cm. NMI increased with temperature at a faster rate than fume weight suggesting that the increases in NMI were related to both quantitative and qualitative changes in the PAC content of the fumes.

Effects of Selected Thai Ancient Remedies Extracts on Mutagenicity and Antimutagenicity Using Ames Test

The extracts of selected Thai household ancient remedies namely: Chantaleela, Homtip-osot, Keaw-hom, Prasachandang, Prasamawaeng, Thoraneesantakat, Tree-hom, Ummaluk-kawatee and Wisampayayai were determined for the mutagenic and antimutagenic effects in the absence of metabolic activation using Salmonella typhimurium TA98 and TA100. Ethanol was the first solvent used to extract the remedies by soxhlet apparatus till exhaustion. Then the marc was further successively extracted with water. It was found that most ethanolic and water extracts of selected Thai ancient remedies were not directly mutagenic, except that the ethanolic extract of Tree-hom exhibited slightly mutagenicity with the mutagenic index of 3.6 and 2.2 on S. typhimurium TA98 and TA100 respectively. However, after treating with nitrite, all extracts showed mutagenic potential on both strains. Antimutagenic assay using 1- aminopyrene-nitrite model revealed that selected Thai ancient remedies could either inhibit or enhance mutagenicity and the concentration-activity relationship was shown. The ethanolic extracts were revealed for more potent activities in mutagenicity and antimutagenicity than water extracts.

Pancreatic cancer risk: associations with meat-derived carcinogen intake in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) cohort.

Epidemiological studies report positive associations between high-temperature cooked meat intake and pancreatic cancer. We assessed associations between dietary intake of heterocyclic amines (HCAs) and benzo(a)pyrene (BaP)-mutagens formed in meat cooked at high temperatures-and incident exocrine pancreatic cancer in a prospective cohort. The 62 581 subjects randomized to screening in the Prostate, Lung, Colorectal, and Ovarian Screening Trial (PLCO) who completed an initial dietary survey that assessed meat intake, cooking methods, and doneness preferences defined the cohort. Subjects were surveyed annually for incident cancers through 2007. A National Cancer Institute research database (CHARRED) was used to estimate HCA and BaP intake and a Mutagenic Activity Index (MAI) from survey data. Proportional hazard ratios (HRs) for risk of pancreatic cancer were estimated from multi-variate Cox regression models by quintile of intake, with the lowest quintile as the referent. During follow-up (median: 10 yr), 248 cases of exocrine pancreatic cancer were confirmed. Preferences for well and very well done meat were generally associated with increased risks. Significant elevations in pancreatic cancer risk were found in upper quintiles of MAI, and individual mutagens 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Compared to the lowest quintile of MAI, the third and fifth quintiles brought HRs of 1.86 (1.22, 2.85) and 1.87 (1.16, 3.02), respectively. These three exposures exhibited significant (P-trend: 0.01-0.03) positive trends in risk as their levels increased Consuming well-done meat cooked at high temperatures, which contains high mutagen levels, appears to confer increased risk of pancreatic cancer.

Effect of oxidation and extent of oxidation on biologically active PACs in asphalt products

Recent studies have reported divergent results in rodent cancer assays using fume condensates from a variety of asphalt products. This paper presents results of a study investigating the role of oxidation, or extent of oxidation, on these findings. Five straight run asphalts, made from widely used crude oils, were used as inputs to both production scale and laboratory oxidation units and processed to a range of softening points used in common roofing products. For each of the five asphalts studied, the oxidation reaction significantly decreased measures of polycyclic aromatic compounds (PACs) that have been linked, previously and in analyses included in this study, to tumor induction in rodent bioassays. Mutagenicity index determined by the modified Ames assay was reduced between 41% and 50% from the input asphalt to the final oxidized product. A fluorescence method tuned to a subset of PAC compounds that have been associated with carcinogenic behavior in mouse bioassays was reduced between 39% and 71%. The decrease was largest in the first quarter of the oxidation reaction. These findings indicate that oxidation, by itself, was not a likely factor in the tumor induction seen in the previous studies. Rather, other factors such as the conditions of fume generation and crude source (coupled with possible differences in distillation endpoints) were more likely to have determined the outcomes. Analyses of previously published data, presented in this paper, suggest that the modified Ames and fluorescence assays are valuable screening tools for use in future health-related asphalt research.

Runoff of genotoxic compounds in river basin sediment under the influence of contaminated soils

Contaminated sites must be analyzed as a source of hazardous compounds in the ecosystem. Contaminant mobility in the environment may affect sources of surface and groundwater, elevating potential risks. This study looked at the genotoxic potential of samples from a contaminated site on the banks of the Taquari River, RS, Brazil, where potential environmental problems had been identified (pentachlorophenol, creosote and hydrosalt CCA). Samplers were installed at the site to investigate the drainage material (water and particulate soil matter) collected after significant rainfall events. Organic extracts of this drained material, sediment river samples of the Taquari River (interstitial water and sediment organic extracts) were evaluated by the Salmonella/microsome assay to detect mutagenicity and by Allium cepa bioassays (interstitial water and whole sediment samples) to detect chromosomal alterations. Positive mutagenicity results in the Salmonella/microsome assay of the material exported from the area indicate that contaminant mixtures may have drained into the Taquari River. This was confirmed by the similarity of mutagenic responses (frameshift indirect mutagens) of organic extracts from soil and river sediment exported from the main area under the influence of the contaminated site. The Allium cepa test showed significant results of cytotoxicity, mutagenic index and chromosome aberration in the area under the same influence. However, it also showed the same similarity in positive results at an upstream site, which probably meant different contaminants. Chemical compounds such as PAHs, PCF and chromium, copper and arsenic were present in the runoff of pollutants characteristically found in the area. The strategy employed using the Salmonella/microsome assay to evaluate effects of complex contaminant mixtures, together with information about the main groups of compounds present, allowed the detection of pollutant dispersion routes from the contaminated site to the Taquari River sediment.

Mutagenicity assessment of contaminated soil in the vicinity of industrial area

In the industrial area of Chinhat, Lucknow (India) wastewater coming from pesticide manufacturing and other industries is used to irrigate the agricultural crops. This practice has been polluting the soil and pollutants might reach the food chain. Gas chromatographic analysis revealed the presence of certain organochlorine pesticides in soil samples. Samples were extracted using different solvents, i.e., hexane, acetonitrile, methanol, chloroform, and acetone (all were HPLC-grade, SRL, India). Soil extracts were assayed for mutagenicity using Ames Salmonella/mammalian microsome test. Mutagenicity was observed in the test samples and TA98 was the most responsive strain for all the soil extracts (irrigated with wastewater) in terms of mutagenic index in the presence (+S9) and absence (-S9) of metabolic activation. In terms of slope (m) of linear dose-response curve for the most responsive strain TA98 exhibited highest sensitivity against the soil extracts in the presence and absence of S9 fraction. Hexane-extracted soil sample (wastewater) exhibited maximum mutagenicity in terms of net revertants per gram of soil in the presence and absence of S9 mix as compared to the other soil extracts. Groundwater-irrigated soil extracts displayed low level of mutagenicity as compared to wastewater-irrigated soil. The soil is accumulating a large number of pollutants due to wastewater irrigation and this practice of accumulation has an adverse impact on soil health.

Abstract 1907: Pancreatic cancer risk update: Associations with meat-derived carcinogen intake in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) cohort

Abstract Epidemiological studies report positive associations between high-temperature cooked meat intake and pancreatic cancer. We assessed the association between dietary intake of heterocyclic amines (HCAs) and benzo(a)pyrene (BaP)_mutagens formed in meat cooked at high temperatures_and incident exocrine pancreatic cancer in a prospective cohort. Methods: The 62,581 subjects randomized to screening in the multi-center Prostate, Lung, Colorectal and Ovarian Screening Trial (PLCO) who completed a dietary survey at study entry, which assessed meat intake, cooking methods, and doneness preferences defined the cohort. Subjects were surveyed annually for incident cancers through 2007; information was also obtained from physicians, next-of-kin, state registries, and national mortality data. A National Cancer Institute research database (CHARRED) was used to estimate intake of HCA, BaP, and mutagenic activity (Mutagenic Activity Index) from the dietary survey data. The proportional hazard ratios (HRs) for risk of pancreatic cancer were estimated from multivariate-adjusted survival analyses. Results: During follow-up (median: 10 yrs), 248 cases of exocrine pancreatic cancer were confirmed. Intake of well- and very-well done meat was generally associated with increased risks. For example, intake of very well done steak yielded an HR (95% confidence interval) of 1.43 (0.80, 2.56). Using estimated mutagen intake, significant elevations in the risk of pancreatic cancer were found in the upper quintiles of the Mutagenic Activity Index, with HRs of 1.81 (1.16, 2.83) and 1.87 (1.16, 3.02) for quintiles 4 and 5, respectively, relative to the lowest quintile. Significantly increased risks were also associated with exposure high levels of two specific mutagens: 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx). The HRs for DiMeIQx for quintiles 4 and 5 were 1.31 (0.86, 1.98) and 1.81 (1.20, 2.74), respectively, while those for MeIQx were 1.34 (0.87, 2.06) and 1.75 (1.11, 2.76). All three of these exposures exhibited significant positive trends in risk as their intake increased (P for linear trend: 0.01-0.03). Conclusion: Additional follow-up of this cohort has provided stronger evidence that consumption of well-done meat with elevated mutagen levels from high temperature cooking confers an increased risk of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1907. doi:10.1158/1538-7445.AM2011-1907

200 The effect of carbohydrate- and fat-restricted diet on B16F10 melanoma model in C57BL6 mice: focus on tumor growth inhibition and its underlying molecular target signaling pathways

Swiss, C57BL6 and CBA males were given 0.1 ml of 40% ethanol per mouse per day for three consecutive days intraperitoneally. These males were mated with untreated virgin Swiss females employing a 4-day mating schedule and three consecutive matings were carried out. In other study, C57BL6 males were given an ascending gradient of 5% to 40% ethanol in drinking water for a total period of 11 weeks. These males were mated with C57BL6 females for 2 weeks. Females were dissected at mid-term pregnancy for the examination of uterine contents including total, live and dead implants. All the investigations comprised at least two or three independent experiments which were evaluated independently as well as after pooling the data. Swiss, C57BL6 and CBA males given 0.1 ml of 40% ethanol, intraperitoneally, gave no evidence of any significant increase in post-implication lethality in the postmeiotic phase of spermatogenesis attributable to ethanol treatment. A moderate but significant reduction in mean total implants indicating pre-implantation losses was seen in Swiss but not in CBA mice. Prolonged feeding of ethanol up to 40% in drinking water failed to provide any evidence of dominant lethal mutations in C57BL6 males at the pre-implantation level and the post-implantation lethals were also not significantly higher than in controls. In Swiss mice, however, the mutagenic index based on both pre- and post-implantation lethality was consistently positive.

Mutagenicity and genotoxicity of tannery effluents used for irrigation at Kanpur, India

The tannery effluents at Kanpur (India) have been in use for irrigation since last many years, polluting soil directly while ground water and food crops indirectly. Gas chromatography–mass spectrometric analysis of the test samples revealed the presence of organic compounds including diisooctyl phthalate, phenyl N-methylcarbamate, dibutyl phthalate, bis 2-methoxyethyl phthalate, and higher alkanes. Tannery effluent extracts were prepared using XAD-4/8 resins, dichloromethane, chloroform, and hexane and tested with Ames Salmonella test and DNA repair-defective Escherichia coli K-12 mutants. In the presence of XAD-concentrated tannery effluent, TA98 found to be the most sensitive strain in terms of mutagenic index followed by TA97a whereas in terms of mutagenic potential TA102 was most responsive. The extracts were also found genotoxic as determined in terms of survival of E. coli K-12 mutants, suggesting the presence of DNA damaging compounds in the tannery effluents. In the light of results, precautious use of tannery effluents for irrigation is suggested.

Evaluation of Pyrogallol-induced Cytotoxicity in Catalase-mutant Escherichia coli and Mutagenicity in Salmonella typhimurium

We evaluated pyrogallol cytotoxicity using Escherichia coli strains that express mammalian catalase gene derived from catalase mutant mice (Cs(b)) and wild-type (Cs(a)), and pyrogallol mutagenicity by Ames test. Pyrogallol was more toxic to Cs(b) rather than to Cs(a) (p < 0.05), while catalase, superoxide dismutase and ascorbic acid decrease the toxic effect. Pyrogallol also showed mutagenic effect (mutagenic index = 3.8 for 10 micromol pyrogallol/plate) while ascorbic acid (19.4% reduction, p < 0.001) and naringin (35.1% reduction, p < 0.001) played a protective role against it. Pyrogallol cytotoxicity and mutagenicity seem to be attributable, at least in part, to reactive oxygen species formation. This study also suggests that newly established catalase mutant E. coli is probably useful in hazard identification of oxidative chemicals.

Genotoxic and Mutagenic Potential of Agricultural Soil Irrigated with Tannery Effluents at Jajmau (Kanpur), India

It is a common practice in India to irrigate agricultural fields with wastewater originating from industries and domestic sources. At Jajmau (Kanpur), India, tannery effluent is used for irrigation purposes. This practice has been polluting the soil directly and groundwater and food crops indirectly. This study is aimed at evaluating the mutagenic impact of soil irrigated with tannery effluent. Soil extracts were prepared using four organic solvents (dichloromethane, methanol, acetonitrile, and acetone) and tested with Ames Salmonella/microsome test and DNA repair-defective E. coli k-12 mutants. Gas Chromatography-mass spectrometric analysis of soil samples revealed the presence of a large number of organic compounds including bis(2-ethylhexyl)phthalate, benzene, 1,3-hexadien-5-yne, 2,4-bis(1,1-dimethyl)phenol, Docosane, 10-methylnonadecane, and many higher alkanes. The soil extracts exhibited significant mutagenicity with Ames tester strains. TA98 was found to be the most sensitive strains to all the soil extracts, producing maximum response in terms of mutagenic index of 14.2 (-S9) and 13.6 (+S9) in the presence of dichloromethane extract. Dichloromethane-extracted soil exhibited a maximum mutagenic potential of 17.3 (-S9) and 20.0 (+S9) revertants/mg soil equivalent in TA100. Methanol, acetonitrile, and acetone extracts were also found to be mutagenic. A significant decline in the survival of DNA repair-defective E. coli K-12 mutants was observed compared to their isogenic wild-type counterparts when treated with different soil extracts. PolA mutant was found to be the most sensitive strain toward all four soil extracts.

Mutagenicity of Mouriri pusa Gardner and Mouriri elliptica Martius

Mouriri pusa Gardner and Mouriri elliptica Martius are fruit-bearing plants of the Melastomataceae family, popularly known in Brazil as puçá-preto or jaboticaba-do-cerrado, and they are used in folk medicine for the treatment of gastric ulcers. In this study, we employ the Ames test to assess the mutagenicity of compounds obtained from the leaves of these species. The methanol extract of the M. pusa was mutagenic to the Salmonella typhimurium strains TA98, TA97a and TA100, with or without metabolic activation. The methanol extract of M. elliptica induced mutagenic activity in TA98 when metabolized with S9 fraction and TA97a with and without S9, but with lower mutagenicity index (MI) and potencies values than those for M. pusa. Enriched fractions of flavonoids and tannins of M. pusa were also evaluated and they demonstrated positive mutagenicity. The highest values of MI and potency were obtained with the flavonoid fraction, which contains large amounts of quercetin, quercetin glycosides and myricetin. These compounds are probably related to the mutagenicity observed in the Ames test. The dichloromethane extract was not mutagenic in any of the test conditions employed.

Examination of the mutagenicity of RDX and its N-nitroso metabolites using the Salmonella reverse mutation assay

The mutagenicity of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and its N-nitroso derivatives hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) were evaluated using the Salmonella tryphimurium reverse mutation assay (Ames assay) with strains TA97a, TA98, TA100, and TA102. Using a preincubation procedure and high S9 activation (9%), RDX was observed to induce weak mutagenesis to strain TA97a with a mutagenicity index (MI) of 1.5–2.0 at a dose range of 32.7–1090 μg/plate. MNX induced moderate mutagenesis to strain TA97a with an MI of 1.6–2.8 at a dose range of 21.7–878 μg/plate. TNX also induced moderate mutagenesis in strain TA97a with an MI of 2.0–3.5 to TA97a at a dose range of 22.7–1120 μg/plate. TNX also caused weak mutagenesis to strain TA100 with S9 activation at the dose of 1200 μg/plate. MNX and TNX induced weak to moderate mutagenesis to strain TA102. Strain TA97a was found to be the most sensitive strain among these four strains. No cytotoxicity of RDX, MNX, and TNX was observed at the concentrations used in this study. Doses were verified by HPLC.

Mutagenic activity in waste from an aluminum products factory in Salmonella/microsome assay

The mutagenic activity of waste material originating from an aluminum products factory was determined by the Salmonella/microsome assay, using the bacterial strains TA100, TA98 and YG1024. The material was obtained by sweeping the factory floor at the end of the work shift. Organic compounds were extracted by ultrasound for 30 min in dichloromethane or 70% ethanol. After evaporation of solvent, these extracts were dissolved in dimethylsulfoxide, and tested for the mutagenic activity at varying concentrations. All the extracts from the factory had mutagenic activity, especially in the YG1024 strain, suggesting the presence of aromatic amines, later confirmed by chemical analysis. The TA98 strain also showed mutagenic activity, though it did not exhibit the highest mutagenicity index observed with the YG1024 strain. In TA100, mutagenic activity was not observed. This study should serve as an alert to management and those who are occupationally exposed, and as a warning that this type of waste should not be discarded in the environment without any control.

Determination of the carcinogenic potential of lubricant base oil using near infrared spectroscopy and chemometrics

In the present study, near-infrared (NIR) spectroscopy and chemometrics techniques have been combined to predict the carcinogenic potential of lubricant base oils. Principal component analysis was used to select the relevant wavelengths of the NIR spectra and principal component regression was employed to develop linear models to predict the total polyaromatic assay (IP 346) and the mutagenicity index (ASTM E1687/95) of lubricant oil samples. The good correlation between NIR normalised spectra (r=0.995; RMSEP=0.4%) and the total polyaromatic assay is discussed. It was concluded that the combination of both techniques could be used for the on-line determination of the carcinogenic potential of lubricant oils.

Mutagenic Potential of Cypermethrin in Mouse Dominant Lethal Assay

Cypermethrin (CYP) is a synthetic pyrethroid insecticide considered to be environmentally safe and widely used in agriculture and veterinary medicine. To ascertain the validity of this opinion, we investigated the mutagenic potential of CYP using the dominant lethal assay in male Swiss albino mice. CYP was administered by gavage at the dose of 20, 40, and 80 mg/kg body weight, dissolved in 0.2 mL corn oil. Treated mice from all groups were mated with untreated virgin females for a period of 6 weeks that covers the entirespermatogenetic cycle. In the pregnant females, we found a high rate of pre- and post-implantation losses. Dominant lethal mutations were induced in a benzo(a)pyrene-treated group (positive control), and a reduction in the number of total implants was found in all CYP-treated groups only during the initial mating weeks. No significant pre-implantation losses were noted in any of the tested doses. However, significant postimplantation losses were identified in the medium and high doses of CYP. A dose-dependent decline in the number of living implants was noticed in all CYP-treated animals during the first 3 weeks, but decreased in the later weeks. The average mutagenic index of 6 weeks was significantly increased only in the high CYP dose. Our results showed that CYP has mutagenic activity, inducing dominant lethal mutations in male germ cells of mice and caution is recommended in the use of this insecticide.

Formation of DNA adducts from oil-derived products analyzed by 32 P-HPLC

The aim of this study was to investigate the genotoxic potential of DNA adducts and to compare DNA adduct levels and patterns in petroleum vacuum distillates, coal tar distillate, bitumen fume condensates, and related substances that have a wide range of boiling temperatures. An in vitro assay was used for DNA adduct analysis with human and rat S-9 liver extract metabolic activation followed by 32P-postlabeling and 32P-high-performance liquid chromatography (32p-HPLC). For petroleum distillates originating from one crude oil there was a correlation between in vitro DNA adduct formation and mutagenic index, which showed an increase with a distillation temperature of 250 degrees C and a peak around a distillation point of approximately 400 degrees C. At higher temperatures, the genotoxicity (DNA adducts and mutagenicity) rapidly declined to very low levels. Different petroleum products showed a more than 100-fold range in DNA adduct formation, with severely hydrotreated base oil and bitumen fume condensates being lowest. Coal tar distillates showed ten times higher levels of DNA adduct formation than the most potent petroleum distillate. A clustered DNA adduct pattern was seen over a wide distillation range after metabolic activation with liver extracts of rat or human origin. These clusters were eluted in a region where alkylated aromatic hydrocarbons could be expected. The DNA adduct patterns were similar for base oil and bitumen fume condensates, whereas coal tar distillates had a wider retention time range of the DNA adducts formed. Reference substances were tested in the same in vitro assay. Two- and three-ringed nonalkylated aromatics were rather low in genotoxicity, but some of the three- to four-ringed alkylated aromatics were very potent inducers of DNA adducts. Compounds with an amino functional group showed a 270-fold higher level of DNA adduct formation than the same structures with a nitro functional group. The most potent DNA adduct inducers of the 16 substances tested were, in increasing order, 9,10-dimethylanthracene, 7,12-dimethylbenz[a]anthracene and 9-vinylanthracene. Metabolic activation with human and rat liver extracts gave rise to the same DNA adduct clusters. When bioactivation with material from different human individuals was used, there was a significant correlation between the CYP 1A1 activity and the capacity to form DNA adducts. This pattern was also confirmed using the CYP 1A1 inhibitor ellipticine. The 32P-HPLC method was shown to be sensitive and reproducible, and it had the capacity to separate DNA adduct-forming substances when applied to a great variety of petroleum products.

Development of an Analytical Marker for the Prediction of Biological Activity of Bitumen Fumes and Bitumens Using the FIA-DMSO Set-Up. Experimental Set-Up and Correlation with Mutagenicity and Carcinogenicity

During the hot application of bitumen-containing materials, as, for instance, in road paving and roofing, fumes are emitted which contain trace amounts of polycyclic aromatic compounds (PACs). The PAC levels are very low, and from the available epidemiological studies, there is no evidence that human exposure to bitumen or its fumes results in any cancer risk to the workforce. For comparative purposes we are trying to identify a laboratory “marker” for bitumen fume condensates (BFCs) which will give a correlation with biological activity and hence carcinogenic potential. Previously it was found that the determination of total 3–6-ring PACs by DMSO extraction followed by gas chromatographic analysis, whose use is well established for mineral oils, showed promise as a marker for BFCs as well. Because standard DMSO extractions require about 4 g of sample, whereas generally only one mg or less of bitumen fume can be sampled, we developed a micro extraction technique, using Flow Injection Analysis (FIA). The FIA-DMSO extraction was coupled with normal phase liquid chromatography (NPLC) followed by gas chromatography with flame ionisation detection. Only 9 μl of sample solution in cyclohexane (at least 2% m/v) is injected into a cyclohexane stream and extracted with DMSO in the FIA coil. A 50 μl cut from the DMSO stream is introduced into a tetrahydrofurane (THF)/n-octane (50/50) stream to the NPLC-column. A 608 μl fraction of the THF/octane stream after the NPLC column, comprising the time window in which the standard EPA 2–6 ring PAC mixture elutes, is directly introduced into the retention gap. After evaporation of most of the solvent in the retention gap, the concentrated analyte is swept into the GC for the quantitative analysis using the FID. The system is selective towards the biologically most active fraction, the unsubstituted and lightly substituted PACs. The 3–6-ring PAC content by FIA-DMSO correlates well with the Mutagenicity Index (MI) of the Mobil modified Ames test (R2= 0.927), these data have been measured on a wide range of materials ranging from bitumen fume condensates, distillate luboils to heavy residual materials such as bitumens. The residual products have to be freed from the heaviest molecules by pentane precipitation. The 3–6-ring PAC content also correlates with carcinogenicity in mouse skin-painting bioassays. This technique enables us to assess the carcinogenic hazard of potential new feedstocks, processing routes and products in an early stage of development.

Mutagenic evaluation of deltamethrin using rodent dominant lethal assay

The dominant lethal test was used to analyse the mutagenic potential of deltamethrin, a synthetic pyrethroid insecticide, in Swiss albino mice. In the treated series, the animals were exposed orally to three different doses (0.36, 0.72 and 1.08 mg/kg body weight) of deltamethrin dissolved in corn oil. Following the treatment, each male of control, as well as of the treated series, was mated with untreated females, every week for a period of 6 weeks. All mated females were sacrificed on the 13th day of separation and their ovaries and uterus were examined. The results revealed that deltamethrin treatment did not impair the mating capacity and fertility of Swiss albino mice. Mutagenic index, pre- and post-implantation losses were assessed. No significant pre-implantation losses were observed either weekly or averagely. Post-implantation losses were observed at medium and high doses of deltamethrin. A slight increase in dominant lethal mutation rate was observed by increasing doses of deltamethrin in early weeks but decreased in later weeks, so an apparent dose response was not observed.