A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra).

The major histocompatibility complex (MHC) acts as an interface between the immune system and infectious diseases. Accurate characterization and genotyping of the extremely variable MHC loci are challenging especially without a reference sequence. We designed a combination of long-range PCR, Illumina short-reads, and Oxford Nanopore MinION long-reads approaches to capture the genetic variation of the MHC II DRB locus in an Italian population of the Alpine chamois (Rupicapra rupicapra). We utilized long-range PCR to generate a 9 Kb fragment of the DRB locus. Amplicons from six different individuals were fragmented, tagged, and simultaneously sequenced with Illumina MiSeq. One of these amplicons was sequenced with the MinION device, which produced long reads covering the entire amplified fragment. A pipeline that combines short and long reads resolved several short tandem repeats and homopolymers and produced a de novo reference, which was then used to map and genotype the short reads from all individuals. The assembled DRB locus showed a high level of polymorphism and the presence of a recombination breakpoint. Our results suggest that an amplicon-based NGS approach coupled with single-molecule MinION nanopore sequencing can efficiently achieve both the assembly and the genotyping of complex genomic regions in multiple individuals in the absence of a reference sequence.

Relaxed selective constraints drove functional modifications in peripheral photoreception of the cavefish P. andruzzii and provide insight into the time of cave colonization.

The genetic basis of phenotypic changes in extreme environments is a key but rather unexplored topic in animal evolution. Here we provide an exemplar case of evolution by relaxed selection in the Somalian cavefish Phreatichthys andruzzii that has evolved in the complete absence of light for at least 2.8 million years. This has resulted in extreme degenerative phenotypes, including complete eye loss and partial degeneration of the circadian clock. We have investigated the molecular evolution of the nonvisual photoreceptor melanopsin opn4m2, whose mutation contributes to the inability of peripheral clocks to respond to light. Our intra- and inter-species analyses suggest that the 'blind' clock in P. andruzzii evolved because of the loss of selective constraints on a trait that was no longer adaptive. Based on this change in selective regime, we estimate that the functional constraint on cavefish opn4m2 was relaxed at ∼5.3 Myr. This implies a long subterranean history, about half in complete isolation from the surface. The visual photoreceptor rhodopsin, expressed in the brain and implicated in photophobic behavior, shows similar evolutionary patterns, suggesting that extreme isolation in darkness led to a general weakening of evolutionary constraints on light-responsive mechanisms. Conversely, the same genes are still conserved in Garra barreimiae, a cavefish from Oman, that independently and more recently colonized subterranean waters and evolved troglomorphic traits. Our results contribute substantially to the open debate on the genetic bases of regressive evolution.

Antiviral activity of mycophenolic acid derivatives in plants.

KEYWORDS: antiviral agents; chemotherapy; polyamidoamine; multivalency.

Genetic structure of Varroa destructor populations infesting Apis mellifera colonies in Argentina.

Although mitochondrial DNA mapping of Varroa destructor revealed the presence of several haplotypes, only two of them (Korean and Japanese haplotypes) were capable to infest Apis mellifera populations. Even though the Korean haplotype is the only one that has been reported in Argentina, these conclusions were based on mites sampled in apiaries from a specific geographical place (Buenos Aires province). To study mites from several sites of Argentina could reveal the presence of the Japanese genotype, especially considering sites near to Brazil, where Japanese haplotype was already detected. The aim of this work was to study the genetic structure of V. destructor populations from apiaries located in various provinces of Argentina, in order to determine the presence of different haplotypes. The study was carried out between January 2006 and December 2009. Phoretic adult Varroa mites were collected from honey bee workers sampled from colonies of A. mellifera located in Entre Ríos, Buenos Aires, Corrientes, Río Negro, Santa Cruz and Neuquén provinces. Twenty female mites from each sampling site were used to carry out the genetic analysis. For DNA extraction a nondestructive method was used. DNA sequences were compared to Korean haplotype (AF106899) and Japanese haplotype (AF106897). All DNA sequences obtained from mite populations sampled in Argentina, share 98% of similitude with Korean Haplotype (AF106899). Taking into account these results, we are able to conclude that Korean haplotype is cosmopolite in Argentina.

Body size variability of Varroa destructor and its role in acaricide tolerance.

Phenotypic plasticity has been defined as the ability of a genotype to produce different phenotypes when exposed to distinct environments throughout its ontogeny. Morphological variability of individuals is an example of this plasticity. Taking into account that several studies have reported a wide morphological variability in Varroa destructor populations, we evaluated if the body size plasticity of the parasite constituted a key factor able to modulate mites survival when they were exposed to a drug bioassays. Drug bioassays against mites were conducted using three different Syzygium aromaticum essential oil concentrations (0.5, 1, and 5 µl/capsule) and controls. After 4 h of exposition, mite mortality was registered. The width (WS) and length (LS) of the dorsal shield were measured in dead mites. General lineal models were carried to determine if V. destructor survival to acaricides was related to the explanatory variables. Data modelling confirmed that WS and LS variables, together with time interaction, were significantly related to V. destructor survival when the parasites were exposed to acaricides. The models proposed demonstrated that for the smaller S. aromaticum essential oil concentration, the larger the parasite body, the greater the probability that it remains alive at the end of the bioassay. Such relationship was inverse for the other two concentrations tested. Possible causes explaining the body size variability in V. destructor individuals were discussed.

Indoor and outdoor air carbonyl compounds correlation elucidated by principal component analysis.

In this study indoor and outdoor air carbonyl compounds concentrations data are processed by means of principal component analysis (PCA). The analysis pointed to the carbonyl compounds sources as well as to their mutual interrelations. A global six sources (components) solution, accounting for the joint variability of the measured variables, was obtained. The existence of a linear function connecting outdoor and indoor components allowed for an exchange model between indoor and outdoor carbonyl compounds to be outlined. The different sources of carbonyl compounds were tentatively identified. Overall, this work demonstrates the presence of a delicate balance between indoor and outdoor carbonyl compounds contamination with the two compartments (indoor and outdoor) engaged in a close relationship.

Dentistry setting and related environment: a preliminary study.

A study was undertaken at the biggest dental hospital in Italy, the "George Eastman Dental Hospital", in order to assess the dentistry environment as it stands with current equipment and dental practice in use. In order to gain information on the possible presence of chemical pollutants from dentistry activity, a simultaneous consecutive 7-day sampling of volatile organic compounds (VOCs) at the dental chair (dentistry indoor) and at the related vicinity outdoor window (dentistry outdoor) was performed. Volatile organic compounds were identified; cyclohexane, benzene, n-eptane, methylcyclohexane, toluene, ethylbenzene, xylenes, trimethylbenzene and methylhexane were quantitatively determined. As a preliminary result, the dentistry indoor (I(D)) and outdoor (O(D)) volatile organic compounds concentrations ratios are always higher than 1 (I(D)/O(D) > 1), pointing to higher VOCs concentrations in dentistry indoor than in outdoor. It should be noted homes indoor/outdoor concentrations ratios (I(D)/O(H) > 1) are usually higher than 1 as well. For an in-depth study, aimed at assessing the level of VOCs in dentistry setting, instantaneous, hourly, daily, and weekly samplings and a greater number of samples are needed.

Analysis of nucleotide diversity of NAT2 coding region reveals homogeneity across Native American populations and high intra-population diversity.

N-acetyltransferase 2 (NAT2), an important enzyme in clinical pharmacology, metabolizes antibiotics such as isoniazid and sulfamethoxazole, and catalyzes the transformation of aromatic and heterocyclic amines from the environment and diet into carcinogenic intermediates. Polymorphisms in NAT2 account for variability in the acetylator phenotype and the pharmacokinetics of metabolized drugs. Native Americans, settled in rural areas and large cities of Latin America, are under-represented in pharmacogenetics studies; therefore, we sequenced the coding region of NAT2 in 456 chromosomes from 13 populations from the Americas, and two from Siberia, detecting nine substitutions and 11 haplotypes. Variants *4 (37%), *5B (23%) and *7B (24%) showed high frequencies. Average frequencies of fast, intermediate and slow acetylators across Native Americans were 18, 56 and 25%, respectively. NAT2 intra-population genetic diversity for Native Americans is higher than East Asians and similar to the rest of the world, and NAT2 variants are homogeneously distributed across native populations of the continent.

[Inhibition of Paenibacillus larvae employing a mixture of essential oils and thymol]. / Inhibición de Paenibacillus larvae empleando una mezcla de aceites esenciales y timol.

In vitro antimicrobial activity of a mixture of two essential oils and thymol against Paenibacillus larvae, causal agent of American Foulbrood (AFB), was evaluated. The essential oils were extracted from cinnamon (Cinnamomum zeylanicum) and thyme (Thymus vulgaris). The third component used, thymol, is the major component of the essential oil of thyme which contains 39.9% of thymol. Minimal inhibitory concentration (MIC) in Mueller-Hinton broth by the tube dilution method and minimal bactericide concentration (MBC) on MYPGP agar were evaluated. Thyme registered MIC values of 150-250 microg/ml and MBC values of 200-300 microg/ml, while the MIC and MBC values obtained for cinnamon were of 50-100 microg/ml and 100-125 microg/ml. Thymol showed similar MIC and MBC values of 100-150 microg/ml. No significant differences between the bacterial strains were detected, but significant differences between essential oils and thymol activity were registered (P<0,01). An inhibitory synergetic effect on AFB was observed reducing MIC and MBC values due to the use of a mixture of 62.5% of thyme, 12.5% of cinnamon and 25% of thymol.

[Microbiological study of dehydrated garlic (Allium sativum L.) and onion (Allium cepa L.)]. / Estudio microbiológico de ajo (Allium sativum L.) y cebolla (Allium cepa L.) deshidratados.

A microbiological study during the process and the storage of garlic (Allium sativum L.) and onion (Allium cepa L.) dehydrated, with the additional barriers of blanching or brine immersion, was made. In all raw materials the average counts of aerobic mesophilic bacteria expressed in CFU/g ranged from 1.2 x 10(2) to 1.6 x 10(3), molds and yeasts from 60 to 1.6 x 10(3), Lactobacillus spp. and Leuconostoc mesenteroides subsp. mesenteroides between 10 and 50. Microorganisms identified were Penicillium spp., Monilia spp., Lactobacillus brevis, Leuconostoc mesenteroides subsp. mesenteroides and yeasts in garlic; Mucor spp., Penicillium spp., Monilia spp., Lactobacillus brevis and yeasts in both types of onions. Leuconostoc mesenteroides subsp. mesenteroides was detected in only kind of onion. In dehydrated garlic storage, Penicillium spp., Monilia spp., Lactobacillus brevis and yeasts were detected. In garlic, when a blanching step was carried out no microflora was detected. Mucor spp., Penicillium spp., Monilia spp. and Lactobacillus brevis were identified in both types of dehydrated onions. When brine immersion was included the microflora detected was significantly lower and only Penicillium spp. were found. The use of additional barriers such as blanching or brine immersion produces an important effect on the microbiological stability in these products.

Estudio microbiológico de ajo(Allium sativum L.) y cebolla (Allium cepa L.) deshidratados / Microbiological study of garlic (Allium sativum L.) and onion (Allium cepaL.) dehydrated

En este trabajo se efectuó un estudio microbiológico durante la deshidratación y el almacenamiento de ajo (Allium sativum L.) y de cebolla (Allium cepa L.). Al ajo se le efectuó un proceso de escaldado y a la cebolla un salmuereado previo a la deshidratación. En las materias primas los recuentos promedio expresados en UFC/g fueron: bacterias aerobias mesófilas entre 1,2 x 102y 1,6 x 103, mohos y levaduras entre 60 y 1,6 x 103, Lactobacillus spp. y Leuconostocmesenteroidessubesp. mesenteroides entre 10 y 50.Se identificaron: Penicillium spp., Monilia spp. Lactobacillus brevis,Leuconostoc mesenteroides subesp. mesenteroides y levaduras en ajo; Mucor spp., Penicillium spp., Monilia spp., Lactobacillus brevis y levaduras en dos tipos de cebolla. Leuconostoc mesenteroides subesp. mesenteroides sólo se detectó en un tipo de cebolla. En ajos deshidratados y durante el almacenamiento se hallaron Penicillium spp., Monilia spp., Lactobacillus brevis y levaduras mientras que los mismos con escaldado no presentaron desarrollo. Mucor spp., Penicillium spp., Monilia spp. y Lactobacillus brevis se identificaron en los dos tipos de cebolla deshidratadas. Con la incorporación del salmuereado la microflora se redujo significativamente hallándose solamente Penicillium spp. La utilización de barreras adicionales de control microbiano tales como escaldado y salmuereado produce un aporte importante a la estabilidad microbiológica de los productos.

A microbiological study during the process and the storage of garlic (Allium sativum L.) and onion (Allium cepa L.) dehydrated, with the additional barriers of blanching or brine immersion, was made. In all raw materials the average counts of aerobic mesophilic bacteria expressed in CFU/g ranged from 1.2 x 102to 1.6 x 103, molds and yeasts from 60 to 1.6 x 103, Lactobacillus spp. and Leuconostocmesenteroides subsp. mesenteroides between 10 and 50. Microorganisms identified were Penicillium spp., Monilia spp., Lactobacillus brevis, Leuconostoc mesenteroides subsp. mesenteroides and yeasts in garlic; Mucor spp., Penicillium spp., Monilia spp., Lactobacillus brevis and yeasts in both types of onions. Leuconostoc mesenteroides subsp. mesenteroides was detected in only kind of onion. In dehydrated garlic storage, Penicillium spp., Monilia spp., Lactobacillus brevis and yeasts were detected. In garlic, when a blanching step was carried out no microflora was detected. Mucor spp., Penicillium spp., Monilia spp. and Lactobacillus brevis were identified in both types of dehydrated onions. When brine immersion was included the microflora detected was significantly lower and only Penicillium spp. were found. The use of additional barriers such as blanching or brine immersion produces an important effect on the microbiological stability in these products.

Estudio microbiológico de ajo (Allium sativum L.) y cebolla (Allium cepa L.) deshidratados. / [Microbiological study of dehydrated garlic (Allium sativum L.) and onion (Allium cepa L.)]

A microbiological study during the process and the storage of garlic (Allium sativum L.) and onion (Allium cepa L.) dehydrated, with the additional barriers of blanching or brine immersion, was made. In all raw materials the average counts of aerobic mesophilic bacteria expressed in CFU/g ranged from 1.2 x 10(2) to 1.6 x 10(3), molds and yeasts from 60 to 1.6 x 10(3), Lactobacillus spp. and Leuconostoc mesenteroides subsp. mesenteroides between 10 and 50. Microorganisms identified were Penicillium spp., Monilia spp., Lactobacillus brevis, Leuconostoc mesenteroides subsp. mesenteroides and yeasts in garlic; Mucor spp., Penicillium spp., Monilia spp., Lactobacillus brevis and yeasts in both types of onions. Leuconostoc mesenteroides subsp. mesenteroides was detected in only kind of onion. In dehydrated garlic storage, Penicillium spp., Monilia spp., Lactobacillus brevis and yeasts were detected. In garlic, when a blanching step was carried out no microflora was detected. Mucor spp., Penicillium spp., Monilia spp. and Lactobacillus brevis were identified in both types of dehydrated onions. When brine immersion was included the microflora detected was significantly lower and only Penicillium spp. were found. The use of additional barriers such as blanching or brine immersion produces an important effect on the microbiological stability in these products.

Mutagens and carcinogens in size-classified air particulates of a northern Italian town.

This research was designed to examine the presence of mutagenic/carcinogenic compounds in urban airborne particulate matter in relation to particles aerodynamic size. Inhalable (< 10 microns) airborne particulate (PM-10) was collected at a low traffic site in an industrialized Northern Italian town, using a high volume sampler equipped with a cascade impactor for particles fractionation. The organic extracts of different fractions were examined for mutagenicity in Salmonella typhimurium strains TA98 and TA98/1,8-DNP6 using the microsuspension procedure, and for polycyclic aromatic hydrocarbons (PAHs) content by gas chromatography. Size fractionated particles were also analysed for heavy metals (Fe, Mn, Zn, Pb, Cu, Cd, Cr, Ni, V) using plasma spectrophotometry. The results of mutagenicity and chemical analyses indicate that, at the site investigated, inhalable particulate was largely made of fine (< 0.5 micron) particles, which accounted for most of PAHs and mutagenicity. A similar pattern of distribution was found for heavy metals, which were relatively more abundant in small (< 1.5 microns) particles compared to coarser ones.

[Exposure to benzene and genotoxic effects among filling station attendants]. / Esposizione a benzene ed effetti genotossici tra gli addetti all'erogazione di carburanti.

Exposure to gasoline vapors is classified by the International Agency for Research on Cancer as possibly carcinogenic to humans, mainly on the basis of the established carcinogenicity of some component chemicals such as benzene. The mechanism of benzene toxicity, particularly its leukemogenic effects, is far from being fully understood. Different studies, aimed at evaluating the risk associated with exposure to benzene through fuels and coordinated by the Istituto Superiore di Sanità, are in progress in Italy. In an environmental monitoring survey on a sample of 111 service stations, conducted in Rome (Italy) in 1992, average yearly personal exposure to benzene, toluene and xylenes were estimated. Chemical determination of benzene and methylbenzene was carried out by GL-gas chromatography. From a sample of 27 service stations 34 fuel samples were collected, and their benzene content was measured by hr-gas chromatography. Subgroups of the filling station attendants undergoing the exposure assessment study, were included in biological monitoring surveys of early indicators of genotoxicity. In particular, 65 subjects were enrolled in a study aimed at evaluating the urinary concentrations of 8-hydroxydeoxyguanosine (8-OHdG), a biological marker of oxidative DNA damage, and 23 filling station attendants were selected for a survey of the frequencies of sister chromatid exchanges (SCE) and micronuclei (MN) in peripheral T lymphocytes. In the exposure assessment survey levels of 0.53, 0.71 e 0.32 mg/m3 in the average yearly personal exposure to benzene, toluene and xylenes, respectively, were estimated (individual means based on 6.5 repeated samples per employee). The daily quantities of super premium gasoline sold proved to be associated with the average yearly personal exposure to benzene, and current smokers showed a significantly lower exposure intensity compared with non-smokers. Among the latter, an increase of 0.11 ln mg/m3 in benzene exposure per unit increase (100 l) in gasoline sold (p < 0.001) was estimated by a multiple regression analysis with some personal characteristics of the subjects included in the model as potentially predictive variables (R2 = 0.17, p (F) < 0.05). Among smokers, however, only the age and the length of employment were able to predict the intensity of benzene exposure. On a sample of 27 filling station attendants, furthermore, the relationship between personal exposure to benzene and benzene fuel content was evaluated and an increase of 0.01 mg/m3 in the personal benzene exposure per unit increase (100 g) in the absolute quantity of benzene in the fuel sold was estimated (p < 0.0001, R2 = 0.50).(ABSTRACT TRUNCATED AT 400 WORDS)

[Exposure to benzene of service station employees and composition of benzene]. / Esposizione a benzene tra gli addetti alle stazioni di rifornimento e composizione della benzina.

The International Agency for Research on Cancer (IARC) classifies gasoline vapours and exhaust fumes from gasoline fueled automobiles as potential human carcinogens. Data on the chemical composition of gasoline marketed in Italy and especially on the concentration of benzene, are rather poor. Within the framework of an investigation aimed at assessing the mean annual level of exposure to aromatic hydrocarbons among gasoline pump attendants, made on a sample of attendants in Rome between December 1991 and November 1992, samples of gasoline were also collected so as to determine the benzene content of the gasoline over the investigation period, assess the variability of benzene concentration in the various gasolines and according to the season of the year, and take account of gasoline composition in analysing the factors determining individual exposure levels of pump attendants. Benzene exposure was measured via gas chromatography of air samples obtained with personal pumps in the breathing zone. The mean benzene exposure level (8 h TWA) of the 27 subjects under study was 1.73 mg/m3 (SD = 5.53). The benzene concentration in the samples of gasoline, which were collected on the same day as personal exposure monitoring was performed, was measured by means of high resolution gas chromatography (hr-GC). Mean benzene levels of 25.03 g/l (SD = 3.47), equivalent to 2.86% by volume, were measured in 24 samples of alkylated gasoline, and mean levels of 23.18 g/l (SD = 3.93), equivalent to 2.65% v/v, were measured in 10 samples of lead-free gasoline. Statistically significant associations were found between individual exposure to benzene and the quantity of gasoline pumped (r = 0.69) and the quantity of benzene present in the gasoline sold on the day monitoring was performed (r = 0.70). Using regression analysis, the estimated increase in the level of personal benzene exposure was 0.01 mg/m3 for every increase of 100 g in the benzene content of the total amount of gasoline sold. This estimation could be used to quantify the expected reduction in benzene exposure levels in service station attendants following a reduction in the benzene content of gasoline.

Chemical and mutagenic evaluation of sludge from a large wastewater treatment plant.

Digested sludges from a wastewater treatment plant were analyzed to assess their level of contamination by some organic (polychlorobiphenyls (PCBs) and chlorinated pesticides) and inorganic (heavy metals) micropollutants and their mutagenicity features. The heavy metal content in none of the samples exceeded the limits set out in EEC Directive 276/86; as far as PCBs are concerned, the sludges analyzed indicated a level of contamination up to two orders of magnitude higher than some Italian agricultural soils. Mutagenicity assays on either crude or fractionated sludge extracts using Salmonella typhimurium tester strains TA98 and TA100 gave negative results, thus suggesting the absence of genotoxic contaminants in the samples investigated.

Exposure assessment in a historical cohort of filling station attendants.

A historical cohort of service station attendants is underway. It is aimed at evaluating possible excess cancer risk in relation to exposure intensity. In this paper we discuss the feasibility of a retrospective exposure assessment by evaluating the association between indicators of workload and the exposure intensity to some aromatic hydrocarbons measured in a sample of current employees. Available for the analysis were 703 personal samples from 111 filling station workers. Measured concentrations of benzene, toluene and xylenes (8-hour time weighted averages) averaged 0.55 mg/m3, 0.71 mg/m3 and 0.32 mg/m3, respectively. The number of vehicles filled, the daily sales of super premium gasoline and motorbike fuel, and the winter season were all significant predictors of the log concentration of benzene in simple regression analyses. The size of the station acted as an effect modifier. While no single variable was able to predict the benzene level in large stations, for small stations an increase of 0.0579 and of 0.0418 in the log benzene concentration per unit increase in super premium gasoline (100 I) and in motorbike fuel (10 I) dispensed, was estimated. The overall variance explained by the multivariate model, however, was only 12.3%. Therefore, a clear categorization of groups with homogeneous and significantly different exposure levels is not achievable. From the point of view of exposure assessment, workers in small stations with higher sales of super premium gasoline tend to have higher exposure levels. This group should be examined in detail when the final results of the cohort study are available.

[Models and strategies for the control of air pollution in large urban areas]. / Modelli e strategia per controllare la contaminazione atmosferica in grandi aree urbane.

In the attempt to define the characteristics of a global approach to the problem of atmospheric pollution, the mathematical and statisticals tools currently available are presented which will allow the rationalization of both the study and the management of reduction policy. An example of a possible application of this approach is given, which presents the results obtained by modelling the data on "conventional" pollutants furnished by the experimental sampling station set up at the Istituto Superiore di Sanità (ISS) in Rome. Since the suggested approach may not do without an integrated management of date, a hypothesis for an information system is outlined as well.

Mutagenicity spectra in bacterial strains of airborne and engine exhaust particulate extracts.

The mutagenicity spectra of the organic extracts of both airborne particulate matter and diesel and gasoline soot particles were determined using a battery of 9 bacterial strains of different genetic specificity. The assays with crude extracts and with fractionated acidic, neutral and basic components revealed striking differences in the patterns of mutagenic responses produced by each of the complex mixtures investigated. The mutagenicity of air particulate matter was shown to depend mainly on direct-acting acidic and neutral compounds, with a lesser contribution of basic promutagens which required exogenous metabolic activation by liver S9. The assays with a diesel soot extract indicated the prevailing contribution of direct-acting acidic and neutral compounds, and suggested an important role also for nitro derivatives other than nitropyrenes. The gasoline exhaust was characterized by powerful promutagenic compounds, belonging to either the acidic, neutral or basic fractions. The implications of these results are discussed with respect to the contribution of engine exhausts to air pollution, and the possible use of mutagenicity spectra in the analysis of environmental complex mixtures.

Further studies on the comutagenic activity of cigarette smoke condensate.

The comutagenic effect exerted by cigarette smoke condensate (CSC) was investigated. In vitro experiments with Salmonella typhimurium strains TA98 and TA98/1.8DNP6 indicated that CSC specifically enhances the mutagenicity of polyaromatic amines such as 2-aminofluorene, 2-acetylaminofluorene, 4-acetylaminofluorene and 2-aminoanthracene. The pattern of comutagenicity of CSC was shown to differ from that of norharman, a tobacco-related known comutagenic substance. Both black and blond tobacco CSCs proved to interact synergistically with 2-aminoanthracene mutagenicity. Chemical fractionation of CSC indicates the occurrence of comutagenic substance(s) in both neutral and basic components. Further in vitro experiments with 2-acetylaminofluorene metabolites and derivatives suggest that the comutagenic effect of CSC could involve later step(s) in the metabolic activation of fluorenylamines, i.e., the conversion of hydroxylamines into ultimate reactive species. The possible occurrence of a synergistic interaction of CSC with chemical mutagens in vivo was evaluated. Administration of 2-aminoanthracene/CSC mixtures, previously shown to be comutagenic in vitro, failed to demonstrate a synergistic effect in SCE induction in bone marrow cells of mice. This apparent discrepancy may rely on divergences in the activation pathways of polycyclic amines in vitro and in vivo.