Copper promotes E. coli laccase-mediated TNT biotransformation and alters the toxicity of TNT metabolites toward Tigriopus japonicus.

Although laccase is involved in the biotransformation of 2,4,6-trinitrotoluene (TNT), little is known regarding the effect of E. coli laccase on TNT biotransformation. In this study, E. coli K12 served as the parental strain to construct a laccase deletion strain and two laccase-overexpressing strains. These E. coli strains were used to investigate the effect of laccase together with copper ions on the efficiency of TNT biotransformation, the variety of TNT biotransformation products generated and the toxicity of the TNT metabolites. The results showed that the laccase level was not relevant to TNT biotransformation in the soluble fraction of the culture medium. Conversely, TNT metabolites varied in the insoluble fraction analyzed by thin-layer chromatography (TLC). The insoluble fraction from the laccase-null strain showed fewer and relatively fainter spots than those detected in the wild-type and laccase-overexpressing strains, indicating that laccase expression levels were interrelated determinants of the varieties and amounts of TNT metabolites produced. In addition, the aquatic invertebrate Tigriopus japonicus was used to assess the toxicity of the TNT metabolites. The toxicity of the TNT metabolite mixture increased when the intracellular laccase level in strains increased or when purified E. coli recombinant Laccase (rLaccase) was added to the culture medium. Thus, our results suggest that laccase activity must be considered when performing microbial TNT remediation.

Enhancement of microbial 2,4,6-trinitrotoluene transformation with increased toxicity by exogenous nutrient amendment.

In this study, the bacterial strain Citrobacter youngae strain E4 was isolated from 2,4,6-trinitrotoluene (TNT)-contaminated soil and used to assess the capacity of TNT transformation with/without exogenous nutrient amendments. C. youngae E4 poorly degraded TNT without an exogenous amino nitrogen source, whereas the addition of an amino nitrogen source considerably increased the efficacy of TNT transformation in a dose-dependent manner. The enhanced TNT transformation of C. youngae E4 was mediated by increased cell growth and up-regulation of TNT nitroreductases, including NemA, NfsA and NfsB. This result indicates that the increase in TNT transformation by C. youngae E4 via nitrogen nutrient stimulation is a cometabolism process. Consistently, TNT transformation was effectively enhanced when C. youngae E4 was subjected to a TNT-contaminated soil slurry in the presence of an exogenous amino nitrogen amendment. Thus, effective enhancement of TNT transformation via the coordinated inoculation of the nutrient-responsive C. youngae E4 and an exogenous nitrogen amendment might be applicable for the remediation of TNT-contaminated soil. Although the TNT transformation was significantly enhanced by C. youngae E4 in concert with biostimulation, the 96-h LC50 value of the TNT transformation product mixture on the aquatic invertebrate Tigriopus japonicas was higher than the LC50 value of TNT alone. Our results suggest that exogenous nutrient amendment can enhance microbial TNT transformation; however, additional detoxification processes may be needed due to the increased toxicity after reduced TNT transformation.

Alterations of biochemical indicators in hepatopancreas of the golden apple snail, Pomacea canaliculata, from paddy fields in Taiwan.

The freshwater golden apple snail, Pomacea canaliculata, is one of the world's 100 worst invasive alien species. The snails' wide distribution, high abundance, and sensitivity to environmental pollution make them a potential bioindicator for environmental contamination. In this study, the biochemical status of golden apple snails collected from paddy fields throughout the island of Taiwan was examined. This study found that the biochemical status of apple snails collected from paddy fields differed from that of animals bred and maintained in the laboratory. Furthermore, certain biochemical endpoints of the snails collected from the paddy fields before and after agricultural activities were also different-hemolymphatic vitellogenin protein was induced in male snail after exposure to estrogen-like chemicals, the hepatic monooxygenase (1.97 +/- 0.50 deltaA(650mm) 30 min(-1) mg(-1) protein in control group) and glutathione S transferase (0.02 +/- 0.01 delta A(340mm) 30 min(-1) mg(-1) protein in control group) snails exposed to pesticides, as well as the hepatopancreatic levels of aspartate aminotransferase (450.00 +/- 59.40 U mg(-1) mg(-1) protein in control group) and alanine aminotransferase (233.27 +/- 42.09 U mg(-1) mg(-1) protein in control group) decreased the indicating that xenobiotics destroyed hepatopancreatic. The above findings reveal that apple snail could be used as a practical bioindicator to monitor anthropogenic environmental pollution.

Differential regulation and activity against oxidative stress of Dps proteins in Bacillus cereus.

In this study, the sequence similarity, structure, ferroxidase activity and efficacy in antagonizing oxidative stress of three Dps-like proteins, Dps1, Dps2 and Dps3, encoded by Bacillus cereus were comparatively analyzed. The three Dps-like proteins are homologous to other bacterial Dps proteins that exhibit ferroxidase activity. Both Dps1 and Dps2 have a typical Dps spherical structure, but Dps3 has a unique filamentous structure. Several dps mutant strains were generated to investigate the functional role of dps genes in cell protection. The dps1 null strain was the most labile to oxidative stress in the stationary phase, and the loss of dps2 resulted in greater sensitivity to peroxide exposure compared with the other mutant strains in the log phase. Interestingly, after simultaneous deletion of dps1 and dps2, the survival rate was dramatically reduced by approximately 5 log in the stationary phase. Immunoblotting analysis demonstrated that Dps1 and Dps2 in the wild-type strain were induced by oxidative stress, and Dps3 responded to general stress in the log phase. Constitutively high expression of Dps2 in a perR null mutant and PerR-specific binding of the promoter region of dps2 confirmed Dps2 as a member of the PerR regulon. In addition, the expression of Dps1 and Dps2, absent any stress, was initiated in the log phase and was abundant in the stationary phase, suggesting that the expression of Dps1 and Dps2 was dependent on the bacterial growth stage. In summary, the three Dps proteins conferred cellular protection, particularly from oxidative stress, and were differentially regulated in response to varied stress conditions.

Female preference for song frequency in the cicada Mogannia formosana Matsumura (Hemiptera: Cicadidae).

In chorusing species, sound frequency has been suggested as a decisive cue for male body size in female mate choice. However, few studies on the female choice of male song frequency have been conducted in cicadas, in which males of most species sing in chorusing groups to attract females for mating. In this study, we investigated female mate choice for song frequency and body size of males of a chorusing cicada, Mogannia formosana, by phonotaxis experiments using two-choice tests and field observation, respectively. The choice proportion of effective responses and the proximity to the stimulus were used to assess the phonotaxis preference. In the field, two types of males (copulating vs. random) were caught, and their body sizes were compared. In phonotaxis experiments, the results revealed that females showed a preference for low-frequency song by approaching the low-frequency stimuli to a significantly greater proximity. By comparing the body sizes of copulating vs. random males, no significant differences were found. However, the copulating males had significantly different body shapes, as expressed by pronotum width regression by body length, from those of random males. We concluded that the sound frequency of male songs and male body shape in M. formosana can be used as cues of mate quality during female mate choice within a chorus. Additionally, we suggest that females of this species might use multiple cues for mate choice in different ranges of communication and that body size might not be the sole criterion of mate quality.

Determinants of establishment success: Comparing alien and native freshwater fishes in Taiwan.

Many parts of Asia, including Taiwan, have suffered severely from freshwater fish invasions. However, few studies using an assemblage approach have been conducted in the region so far that would help to prioritize suitable preventive actions. In this study, we focused on the invasion process from the import stage to the establishment stage, and defined establishment success as the success during this predefined process. We used datasets of freshwater fish assemblages in Taiwan to (1) compare established versus non-established alien species to distinguish the determinants of establishment success, and (2) to use these determinants to test a life history hypothesis which predicts that the magnitudes of the determinants should be significantly different between established alien species and native species. We collated a dataset for freshwater fish species which were imported into Taiwan (n = 118) of which some successfully established (n = 26), and another dataset for freshwater fish species native to Taiwan (n = 77). For each imported species, we collected data for 17 variables, including two phylogenetic, two human-use, two invasion history, and 11 life history variables. We then used decision tree methods, which have advantages in analyzing datasets with many variables of mixed types without the need to make assumptions about data distributions and input data for missing values. Our results showed that aquaculture use and maximum body length were the most important determinants for predicting establishment success of alien freshwater fish in Taiwan. Comparing five important determinants between established alien versus native species showed that the established alien species were significantly more often used in aquaculture, were associated with a higher number of established countries, and had a larger body length and greater highest water temperature tolerance than the native species. We thus conclude that our results provided evidence to support our stated hypothesis. We suggest that aquaculture use, measures of body size, and the number of previously invaded countries may alert researchers and conservation managers to species with a high establishment potential, especially for countries with similar conditions as those in Taiwan.

Acoustic preadaptation to transmit vocal individuality of savanna nightjars in noisy urban environments.

As urbanization has expanded dramatically, the impacts of urban noise on wildlife have drawn increasing attention. However, previous studies have focused primarily on diurnal songbirds and much less on nocturnal nonpasserines such as nightjars. The savanna nightjar has recently successfully colonized urban areas in Taiwan. Using 1925 calls recorded from 67 individuals, we first investigated the individual differences of the acoustic structures; and, for those acoustic variables with significant individual differences, we examined the correlation between the acoustic structures and the ambient noise levels. We then compared the transmission efficacy of vocal individuality among three sets of acoustic variables: all acoustic variables, noise-related variables, and noise-unrelated variables. Using seven artificial frequency-shifted calls to represent seven different individuals in playback-recording experiments, we also investigated the transmission efficacy of vocal individuality and variable accuracy in three different urban noise levels (high, medium, low). We found that all 30 acoustic variables derived from the acoustic structures demonstrated significant individual differences, and 14 frequency-based variables were negatively correlated with ambient noise levels. Although transmission efficacy was significantly affected by urban noise, individuality information was still transmitted with high accuracy. Furthermore, the noise-unrelated structures (which included the maximum frequency, the maximum amplitude frequency, and the mean frequency of the call) had a significantly higher transmission efficacy of vocal individuality than the noise-related variables (which included the minimum frequency, the frequency at the start and the end of the call) in both field observation and playback-recording experiments. We conclude that these noise-unrelated acoustic features may be one of the key preadaptations for this nocturnal nonpasserine to thrive so successfully in its newly adopted urban environment.

orf4 of the Bacillus cereus sigB gene cluster encodes a general stress-inducible Dps-like bacterioferritin.

The function of orf4 in the sigB cluster in Bacillus cereus ATCC 14579 remains to be explored. Amino-acid sequence analysis has revealed that Orf4 is homologous with bacterioferritins and Dps. In this study, we generated an orf4-null mutant and produced recombinant protein rOrf4 to establish the role of orf4. In vitro, the purified rOrf4 was found to exist in two distinct forms, a dimeric form and a polymer form, through size exclusion analysis. The latter form exhibited a unique filament structure, in contrast to the typical spherical tetracosamer structure of bacterioferritins; the former can be induced to form rOrf4 polymers immediately after the addition of FeCl(2). Catalysis of the oxidation of ferrous irons by ferroxidase activity was detected with rOrf4, and the mineralized irons were subsequently sequestered only in the rOrf4 polymer. Moreover, rOrf4 exerted DNA-protective activity against oxidative damage via DNA binding in a nonspecific manner, as is seen with Dps. In vivo, deletion of orf4 had no effect on activation of the alternative sigma factor sigma(B), and therefore, orf4 is not associated with sigma(B) regulation; however, orf4 can be significantly upregulated upon environmental stress but not H(2)O(2) treatment. B. cereus strains with constitutive Orf4 expression exhibited a viability higher than that of the orf4-null mutant, under specific oxidative stress or heat shock. Taken together, these results suggest that Orf4 functions as a Dps-like bacterioferritin in response to environmental stress and can provide cell protection from oxidative damage through iron sequestration and DNA binding.

Male dimorphism in three sympatric species of Onthophagus (Coleoptera: Scarabaeidae).

Onthophagus trituber, O. taurinus, and O. proletarius, three sympatric dung beetle species native to southern Taiwan, exhibit normally distributed body size (pronotum width) but non-normally distributed horn length due to the presence of horned and hornless morphs. The scaling relationships between horn length and body size were established by using sigmoidal and segmented linear estimation, and the horned/hornless ratio in each of the species was estimated. The ratios estimated by sigmoidal curves showed that all three species were biased toward the hornless morph, whereas the ratios from segmented lines showed that only one species (O. taurinus) was biased toward the hornless morph. Nevertheless, the results of the two methods of estimation were concordant in ranking the horned morph proportions among the three species; O. proletarius had the largest proportion of horned morphs, whereas O. taurinus had the smallest, suggesting that the fewer horned morph in O. taurinus likely resulted from both intra- and inter-specific competition.

Contrasting determinants for the introduction and establishment success of exotic birds in Taiwan using decision trees models.

BACKGROUND: Biological invasions have become a major threat to biodiversity, and identifying determinants underlying success at different stages of the invasion process is essential for both prevention management and testing ecological theories. To investigate variables associated with different stages of the invasion process in a local region such as Taiwan, potential problems using traditional parametric analyses include too many variables of different data types (nominal, ordinal, and interval) and a relatively small data set with too many missing values. METHODS: We therefore used five decision tree models instead and compared their performance. Our dataset contains 283 exotic bird species which were transported to Taiwan; of these 283 species, 95 species escaped to the field successfully (introduction success); of these 95 introduced species, 36 species reproduced in the field of Taiwan successfully (establishment success). For each species, we collected 22 variables associated with human selectivity and species traits which may determine success during the introduction stage and establishment stage. For each decision tree model, we performed three variable treatments: (I) including all 22 variables, (II) excluding nominal variables, and (III) excluding nominal variables and replacing ordinal values with binary ones. Five performance measures were used to compare models, namely, area under the receiver operating characteristic curve (AUROC), specificity, precision, recall, and accuracy. RESULTS: The gradient boosting models performed best overall among the five decision tree models for both introduction and establishment success and across variable treatments. The most important variables for predicting introduction success were the bird family, the number of invaded countries, and variables associated with environmental adaptation, whereas the most important variables for predicting establishment success were the number of invaded countries and variables associated with reproduction. DISCUSSION: Our final optimal models achieved relatively high performance values, and we discuss differences in performance with regard to sample size and variable treatments. Our results showed that, for both the establishment model and introduction model, the number of invaded countries was the most important or second most important determinant, respectively. Therefore, we suggest that future success for introduction and establishment of exotic birds may be gauged by simply looking at previous success in invading other countries. Finally, we found that species traits related to reproduction were more important in establishment models than in introduction models; importantly, these determinants were not averaged but either minimum or maximum values of species traits. Therefore, we suggest that in addition to averaged values, reproductive potential represented by minimum and maximum values of species traits should be considered in invasion studies.

Interspecific comparison of traffic noise effects on dove coo transmission in urban environments.

Most previous studies concerning avian adaptation to anthropogenic noise have focused on songbirds, but few have focused on non-songbirds commonly found in urban environments such as doves. We conducted field playback-recording experiments on the perch-coos of five dove species, including four native Taiwan species (the spotted dove, Spilopelia chinensis, the oriental turtle-dove, Streptopelia orientalis, the red collared-dove, Streptopelia tranquebarica, and the emerald dove, Chalcophaps indica) and one species not native to Taiwan (the zebra dove, Geopelia striata) to evaluate the detection and recognition of dove coos in habitats with differing levels of traffic noise. Our results suggest that traffic noise has selected dominant urban species such as the spotted dove to temporally and spatially adjust cooing to reduce the masking effects of traffic noise and rare urban species such as the emerald dove to avoid areas of high traffic noise. Additionally, although the zebra dove had the highest coo frequency among the study species, its coos showed the highest detection value but not the highest recognition value. We conclude that traffic noise is an important factor in shaping the distribution of rare and dominant dove species in urban environments through its significant effects on coo transmission.

Growth, Diet Composition and Reproductive Biology of the Invasive Freshwater Fish Chevron snakehead Channa striata on a Subtropical Island.

Kuan-chung Li, Bao-sen Shieh, Yuh-wen Chiu, Da-ji Huang, and Shih-hsiung Liang (2016) The Chevron snakehead (Channa striata) has been invading Taiwan for over 30 years, and it is currently widely distributed across diverse aquatic habitats within the island. Due to its strong environmental adaptability and carnivorous diet, C. striata has caused great negative impacts to the biodiversity of native fishes and aquatic organisms in Taiwan. To effectively restrain its spatial distribution and population, the objective of this study was to investigate the growing conditions, diet composition, and reproductive biology of C. striata in the field. In total, 294 individuals were collected from wetlands, irrigation canals, streams, and reservoirs in southern Taiwan from September 2008 to December 2010. Among 272 sex-identified individuals, more females (164) were collected than males (108). The morphological differences between the sexes could not be distinguished by the 10 body measurements recorded. Diverse food items, including snails, odonates, fishes, amphibians, and reptiles, were identified in the stomachs of 35 individuals. The minimum body length of sexually mature C. striata females exhibited at a standard length of 24.5 cm (total length 28 cm). The appearance of mature oocytes were mainly observed from July to November in 2009 and from April to October in 2010. Greater absolute fecundity (oocyte/individual) was estimated in Taiwan for C. striata than in its original distribution range possibly due to less water level fluctuation in the sampling habitats of Taiwan. The relative fecundity (oocyte/g) for C. striata was considered lower but within the documented range in Taiwan when compared with its original habitat in Malaysia. To effectively manage C. striata in Taiwan, regionally eradiating young and adult individuals, especially during the reproductive season and educating people to stop releasing it in the wild are possible ways to restrain and control the further spread of this exotic fish in Taiwan.

Acoustic and temporal partitioning of cicada assemblages in city and mountain environments.

Comparing adaptations to noisy city environments with those to natural mountain environments on the community level can provide significant insights that allow an understanding of the impact of anthropogenic noise on invertebrates that employ loud calling songs for mate attraction, especially when each species has its distinct song, as in the case of cicadas. In this study, we investigated the partitioning strategy of cicada assemblages in city and mountain environments by comparing the acoustic features and calling activity patterns of each species, recorded using automated digital recording systems. Our comparison of activity patterns of seasonal and diel calling revealed that there was no significant temporal partitioning of cicada assemblages in either environment. In addition, there was no correlation between the acoustic distance based on spectral features and temporal segregation. Heterospecific spectral overlap was low in both city and mountain environments, although city and mountain cicada assemblages were subject to significantly different levels of anthropogenic or interspecific noise. Furthermore, for the common species found in both environments, the calling activity patterns at both seasonal and diel time scales were significantly consistent across sites and across environments. We suggest that the temporal calling activity is constrained by endogenous factors for each species and is less flexible in response to external factors, such as anthropogenic noise. As a result, cicada assemblages in city environments with low species diversity do not demonstrate a more significant temporal partitioning than those in mountain environments with high species diversity.

Airborne Transmission of Melioidosis to Humans from Environmental Aerosols Contaminated with B. pseudomallei.

Melioidosis results from an infection with the soil-borne pathogen Burkholderia pseudomallei, and cases of melioidosis usually cluster after rains or a typhoon. In an endemic area of Taiwan, B. pseudomallei is primarily geographically distributed in cropped fields in the northwest of this area, whereas melioidosis cases are distributed in a densely populated district in the southeast. We hypothesized that contaminated cropped fields generated aerosols contaminated with B. pseudomallei, which were carried by a northwesterly wind to the densely populated southeastern district. We collected soil and aerosol samples from a 72 km2 area of land, including the melioidosis-clustered area and its surroundings. Aerosols that contained B. pseudomallei-specific TTSS (type III secretion system) ORF2 DNA were well distributed in the endemic area but were rare in the surrounding areas during the rainy season. The concentration of this specific DNA in aerosols was positively correlated with the incidence of melioidosis and the appearance of a northwesterly wind. Moreover, the isolation rate in the superficial layers of the contaminated cropped field in the northwest was correlated with PCR positivity for aerosols collected from the southeast over a 2-year period. According to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analyses, PFGE Type Ia (ST58) was the predominant pattern linking the molecular association among soil, aerosol and human isolates. Thus, the airborne transmission of melioidosis moves from the contaminated soil to aerosols and/or to humans in this endemic area.

Application of real-time PCR, DGGE fingerprinting, and culture-based method to evaluate the effectiveness of intrinsic bioremediation on the control of petroleum-hydrocarbon plume.

Real-time polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and the culture-based method were applied in the intrinsic bioremediation study at a petroleum-hydrocarbon contaminated site. The genes of phenol hydroxylase (PHE), ring-hydroxylating toluene monooxygenase (RMO), naphthalene dioxygenase (NAH), toluene monooxygenase (TOL), toluene dioxygenase (TOD), and biphenyl dioxygenase (BPH4) were quantified by real-time PCR. Results show that PHE gene was detected in groundwater contaminated with benzene, toluene, ethylbenzene, xylene isomers (BTEX) and methyl tert-butyl ether (MTBE), and this indicates that intrinsic bioremediation occurred at this contaminated site. Results from DGGE analyses reveal that the petroleum-hydrocarbon plume caused the variation in microbial communities. In this study, MTBE degraders including Pseudomonas sp. NKNU01, Bacillus sp. NKNU01, Klebsiella sp. NKNU01, Enterobacter sp. NKNU01, and Enterobacter sp. NKNU02 were isolated from the contaminated groundwater using the cultured-based method. Results from MTBE biodegradation experiment show that the isolated bacteria were affected by propane. This indicates that propane may influence the metabolic pathway of MTBE by these bacteria. Knowledge and comprehension obtained from this study will be helpful in evaluating the occurrence and effectiveness of intrinsic bioremediation on the remediation of petroleum-hydrocarbon contaminated groundwater.

Impact of cadmium on the bacterial communities in the gut of Metaphire posthuma.

The effects of cadmium (Cd) contamination in soil onto the bacterial communities of the guts pooled from ten Metaphire posthuma were addressed during 14 days' incubation. We found that about 50% of Cd (5mg/kg, dry weight soil) in the contaminated soil was bio-accumulated into the earthworms. DNA was extracted from the guts of M. posthuma and their dwelling soil irrespective of Cd treatment for the analysis of the bacterial communities of guts in M. posthuma and in soil by PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis). A distinctive cluster of bacterial communities of the guts in the earthworm with and without Cd treatment using the analysis of unweighted pair-group method using arithmetic averages (UPGMA) was observed, indicating that the bacterial community of guts could be changed by Cd. However, no differences in the bacterial communities in soil irrespective of Cd treatment were observed, which could be resulted from the bioremediation of Cd by earthworms leading to insignificant effect of Cd on bacterial communities in soil. For the sequencing of some of the dominant bands in the DGGE profile, Bradyrhizobium japonicum, Stenotrophomonas sp. D2, and Labrys, sp. CC-BB4, whose sequences display an identity of more than 97% using blast program against a known sequence in the GeneBank database and Ribosomal database, were identified. Collectively, our results showed that earthworm treatment can decrease the concentrations of Cd in soil, and Cd cause a shift in the bacterial communities in the guts of M. posthuma. The application of M. posthuma for Cd bioremediation would be desired.

Cloning, expression, and characterization of cadmium-induced metallothionein-2 from the earthworms Metaphire posthuma and Polypheretima elongata.

In this study we report the sequences of MT-2 cDNA from two species of Megascoleidae earthworms, Metaphire posthuma and Polypheretima elongata, by mRNA differential display after exposure of the organisms to cadmium. Complementary (c)DNA was verified as the MT-2 gene by the characteristics of its predicted translation product, namely a high cysteine content, conserved CXC motifs, and a molecular weight of around 8 kDa. Amino acid sequence alignment revealed a conserved TKCCG in the cloned MT-2 of both megascolecid earthworms instead of the corresponding conserved TQCCG found in lumbricid earthworms. The cDNAs corresponding to the two megascolecid MT-2 genes were expressed, and the MT-2 proteins were purified for biochemical characterization. The binding of Cu2+ exhibited monophasic kinetics and those of Zn2+ and Cd2+ biphasic kinetics. The proteins bound more tightly to Cd2+ than to Zn2+ and more tightly still to Cu2+. Zn-MT and apo-MT were the most effective at scavenging free radicals, followed by Cd-MT. In conclusion, MT-2s from M. posthuma and P. elongata showed unique sequence features compared to those of lumbricid earthworms. These earthworms could be used to evaluate heavy-metal pollution in soil due to the inducible MT-2 by cadmium exposure.

Single strand dna breaks in human lymphocytes exposed to para-phenylenediamine and its derivatives.

Para-Phenylenediamine (PPD), the main aromatic amines used in the hair dye formation, and its four derivatives (2-chloro-p-phenylenediamine, 4-chloro-o-phenylenediamine, 2-nitro-p-phenylenediamine, and 4-nitro-o-phenylenediamine) were examined for their potential to produce single strand DNA breaks in human lymphocytes using the alkaline comet assay. Results revealed that all the tested chemicals within the range of doses from 100 microM to 500 microM showed the genotoxicity in a dose-dependent manner after the incubation of lymphocytes with these chemicals for 2 h. In this study, we first reported that PPD and its four derivatives can elicit the type of single strand breaks in human lymphocytes.