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Biomedical subjects

J Nath

Publications and source records attributed to J Nath.

At least 37 records · Page 2Linked to original sources

Chromosome aberrations of clonal origin in irradiated and unexposed individuals: assessment and implications.

Chromosome painting has proven useful for the detection of chromosomal rearrangements, although the presence of cells containing clonal aberrations can have an effect on the outcome of cytogenetic analyses (e.g. aberration frequency and chromosomal distribution studies). Cells with clonal chromosomal changes have been found in studies of both radiation-exposed Chernobyl cleanup workers ("liquidators") and healthy unexposed human subjects. We have used a simple statistical method to aid in the identification of individuals from distinct Chernobyl radiation-exposed and unexposed control populations who may possess cells containing clonal rearrangements. A chi2 value determined from the observed number of aberrations and the expected number based on chromosome length that corresponds to a probability less than 0.005 appears to be an indicator of clonality. These selected individuals can be analyzed further for clonality, thereby sparing detailed examination of the entire population. Here we present an analysis of individuals possessing clonal aberrations to assess the influence of clonality on the results of cytogenetic studies. Our results show that the subtraction of clonal events from the chi2 calculation for the "outliers" results in nearly all of these values losing their statistically significant deviation from proportionality. These adjustments can also be made to prevent the overestimation of frequencies of chromosome aberrations for biodosimetry. The frequency of clonal aberrations appears to increase as a function of age in control subjects, whereas an age effect was not evident in Chernobyl liquidators. This suggests that spontaneous and radiation-induced clonal expansion are occurring in control subjects and liquidators, respectively.

Adult↗

In vitro and in vivo transformation in rat tracheal epithelial cells exposed to diesel emission particles and related compounds.

The rat tracheal epithelial (RTE) cell transformation assay was performed to determine the transforming activity of diesel emission particles (DEPs) and two related compounds, 1-nitropyrene (1-NP) and dibenzo(a,i)pyrene (DBP). RTE cells were treated with these agents in vitro and in vivo. Transformed cells from foci induced by these agents were passaged over 20 times to establish immortal cell lines. Results show that (1) DEPs- and 1-NP-induced cell transformation only with the in vivo exposure (30-75 mg/kg bw DEPs and 15-60 mg/kg bw 1-NP); (2) positive dose-related responses to DBP were found with both in vitro (0.05-0.50 microg/ml) and in vivo (7.5-30 mg/kg bw) exposures; (3) the fraction of transformed foci becoming cell lines was in the order of 1-NP(25/48) > DBP(8/28) > DEPs(0/30). These results indicate that (1) DEPs, 1-NP and DBP are capable of transforming rat tracheal epithelial cells, however, the transforming activity of DEPs and -NP may be dependent on metabolic activation, and (2) transformed cells induced by DEPs have a very low probability, if any, of becoming cell lines.

Animals↗

Protective effect of vanillin on radiation-induced micronuclei and chromosomal aberrations in V79 cells.

Vanillin (VA), an anticlastogen, has been demonstrated to inhibit gene mutations in both bacterial and mammalian cells. However, the data on its effect against radiation-induced cytogenetic damage are limited. The aim of this study was to investigate the protective effect of VA on radiation-induced chromosomal damage in V79 cells. Exponentially growing cells were exposed to five doses of X-rays (1-12 Gy) and UV radiation (50-800 microJ x 10(2) cm-2 and posttreated with 3 concentrations of VA (5, 50 or 100 micrograms ml-1 for 16 h for micronucleus (MN) and 18 h for structural chromosomal aberration (SCA) analyses. MN and SCA assays were performed concurrently according to standard procedures. Results indicate that there was a dose related increase in the percent of micronucleated binucleated cells (MNBN) (5.6 to 79.6) and percent of aberrant cells (Abs) (12 to 98) with X-ray treatment alone. Inhibition studies showed that the addition of VA at 100 micrograms ml-1 significantly reduced the percent of MNBN (21 to 48) induced by X-ray at 1, 2, and 4 Gy. There was a slight decrease in percent MNBN at 5 and 50 micrograms VA ml-1. All three concentrations of VA decreased percent Abs (15.7 to 57.1) induced by X-rays at all doses. UV radiation alone significantly increased percent MNBN (3.5 to 14.8) and percent Abs (17 to 29). Addition of 50 or 100 micrograms VA ml-1, significantly decreased percent MNBN (31.7 to 86.2) and percent Abs (54.5 to 90.9) at all doses of UV radiation. A decrease in percent MNBN (2.8 to 72.4) and percent Abs (34.8 to 66.7) was also noted at 5 micrograms VA ml-1. These data clearly indicate the protective effect of VA on radiation-induced chromosomal damage, suggesting that VA is an anticlastogenic agent.

Animals↗

Inhibition of methotrexate-induced chromosomal damage by folinic acid in V79 cells.

Methotrexate (MTX), an anticancer compound, is widely used in the treatment of leukemia. It induces cytogenetic damage as well as cytostatic effects on a variety of cell systems. Folinic acid (Leucovorin) is generally administered along with MTX as a rescue agent to decrease MTX-induced toxicity. However, information regarding the inhibitory effect of folinic acid against cytogenetic damage caused by MTX is limited. This study was conducted to assess the cytogenetic effect of MTX and its inhibition by folinic acid (FA) using the micronucleus and chromosomal aberration assays concurrently. Exponentially growing V79 cells were treated with MTX at five different concentrations (5-100 micrograms ml-1) with S9 microsomal fraction for 6 h and post-treated with two concentrations of FA (5 or 50 micrograms) for 40 h. Results indicate that MTX alone induced a concentration-related increase in % micronucleated binucleated cells (MNBN) and % aberrant cells (Abs). There was a decrease in nuclear division index (NDI) with increase in MTX concentration. Similarly, the mitotic index (MI) also decreased in all concentrations of MTX tested. The addition of FA at 50 micrograms ml-1 significantly reduced % MNBN (40-68%) and % Abs (36-77%). Inhibition was also seen at 5 micrograms FA (12 to 54% for MNBN and 20 to 61% for Abs). These results indicate that FA is capable of reducing the cytogenetic damage induced by MTX and appears to be an anticlastogenic agent.

Animals↗

Interactive role of nitric oxide and superoxide anion in neutrophil-mediated endothelial cell injury.

This study addresses the interactive role of nitric oxide (NO) and reactive oxygen intermediates (ROI) by direct quantitation of NO and superoxide (O2-) in human neutrophil (PMN)-endothelial cell (EC) co-culture during PMN-mediated EC injury. The results directly demonstrate an inverse correlation between NO and ROI levels in PMN-EC co-culture, which significantly alters the PMN-EC adhesion and PMN-mediated EC killing. N-formylmethionyl-leucyl-phenylalanine (fMLF)-stimulated PMN adhesion to cytokine-treated EC was decreased (> 25%) in the presence of S-nitroso-N-penicillamine, a NO donor. NO also inhibited EC killing by stimulated PMN, suggesting its cytoprotective role. In addition, a significant decrease in NO levels was observed in the PMN-EC co-culture compared with the EC cultured alone (422.45 +/- 35.76 vs. 800.79 +/- 41.69 pmol). The reduced NO levels were restored by the addition of superoxide dismutase, a scavenger of O2-, suggesting that PMN-derived O2- is involved in the neutralization of NO in the co-culture. The results indicate an inverse correlation between NO and O2- in PMN-EC interactions and suggest the need for a critical balance between these two radicals in the regulation of PMN-mediated tissue injury.

Cell Adhesion↗

Biochemical hemodynamic and hematological changes during transcervical resection of the endometrium using 1.5% glycine as the irrigating solution.

OBJECTIVE: To study fluid absorption during transcervical resection of the endometrium (TCRE) and its effect on the biochemical, hemodynamic and hematological alterations so that life threatening complications of fluid overload may be prevented. METHOD: Intraoperative fluid (1.5% glycine) absorption in 46 women undergoing TCRE was studied and correlated using biochemical parameters (serum sodium, potassium, total proteins, creatinine and blood urea), hemodynamic parameters (pulse rate, blood pressure, oxygen saturation and end tidal CO2) and hematological parameters. Twenty five of these patients had received danazol (800 mg/day) for six weeks prior to TCRE. RESULT: The mean glycine deficit during TCRE was found to be 474.45 ml, with a mean total inflow of 3802.17 ml. Amongst all of the parameters, only serum sodium levels were found to be significantly inversely correlated with the glycine deficit. No case of hyponatremia occurred below a deficit of 1000 ml. Severe hyponatremia was reported in three cases (6.4%) and all three had a glycine deficit of more than 1000 ml. No case of pulmonary edema was noted. The mean glycine deficit was significantly lower (P=0.007) and the duration of procedure significantly shorter (P=0.0009) in the patients who had received danazol. None of the patients in the danazol group had fluid absorption of more than 1000 ml. CONCLUSION: Close monitoring of fluid inflow and outflow should be done during TCRE. Above a deficit of 1000 ml, serum sodium should be measured to detect significant hyponatremia. The use of danazol for endometrial preparation also reduces the mean amount of fluid absorbed.

Blood Proteins↗

Frequency, distribution and clonality of chromosome damage in human lymphocytes by multi-color FISH.

Whole chromosome painting was used to determine whether the use of different sets of paints would influence results obtained from the analysis of human peripheral blood lymphocytes from 27 healthy unexposed subjects. Painting was also used to determine if aberration frequencies are proportional to the size of selected chromosomes in human lymphocytes irradiated in vitro. The in vitro results showed that the frequencies of radiation-induced stable aberrations (i.e. translocations and insertions) in chromosomes 3, 5 and 6 painted in unique colors are proportional to chromosome size. Aberration frequencies in the normal subjects were measured using two different sets of paints, one set for chromosomes 3, 5 and 6 where each chromosome was labeled in a unique color and one set where chromosomes 1, 2 and 4 were painted in a single color. The frequency of aberrations among chromosomes 1-6 in the population as a whole was also found to be proportional to chromosome size. However, some individual subjects had a distribution of damage that was not proportional to chromosome size due to the presence of clones of abnormal cells. Aberration frequencies measured in chromosomes 3, 5 and 6 as a set were highly correlated with those observed in chromosomes 1, 2 and 4 as a set, after adjusting for the different amounts of the genome that were painted. We also determined whether differences exist in the aberration frequencies measured by two scoring systems: the classical method, where reciprocal exchanges are scored as single events, and PAINT, where each break junction is scored as a single event. The two scoring systems gave highly correlated results for translocations and differed by a constant value (PAINT x 0.58 = classical method). Approximately 27% of translocations were observed to be non-reciprocal due to a failure to detect exchanges involving small amounts of material or to a non-reciprocal exchange mechanism. Our results support the hypothesis that cytogenetic evaluations for biodosimetry can be performed with any one or more of the chromosomes studied here and indicate that the aberration frequency measurements are independent of the scoring system selected for the evaluation. We also present a simple statistical method for identifying subjects that may possess clonal aberrations.

Adolescent↗

Safety screening of drugs in cancer therapy.

Development of new drugs requires a thorough investigation of efficacy and safety of pharmaceuticals. The potential risks and benefits of drugs used in chemotherapy are carefully considered such that the benefits of using a new drug outweigh the risks in terms of the side effects caused by the drug. Damage to normal cells, tissues, organs and/or the whole organism is a big concern. Several tests are now routinely performed and are required for drug approval by various regulatory agencies around the globe. The primary goals of such preclinical safety evaluation of drugs are: (1) to identify an initial safe starting dose and subsequent dose escalation scheme to humans; (2) to identify potential target organs of toxicity and reversibility of toxicity; (3) to identify potential damage to the genetic material (genotoxicity); and (4) to identify parameters of clinical monitoring. In this paper, various models for genotoxicity assays are presented. These include: Ames assay, in vitro chromosome aberration assay and an in vivo micronucleus assay. New technologies, such as DNA adduct formation, DNA strand breakage, apoptotic changes, p53 gene expression and transgenic animal models, are also considered.

Aneuploidy↗

Fluorescence in situ hybridization (FISH): DNA probe production and hybridization criteria.

We describe methods for the production of fluorescence in situ hybridization (FISH) probes and the utilization of these probes for the detection of complementary DNA sequences with accuracy and sensitivity for application in both basic research and clinical diagnosis. Due to the frequent use of FISH in many laboratories, it is important to apply the most convenient and reproducible approach. This review describes some of the most recent techniques, and includes versatile, effective and simple methods of probe production and fluorescence in situ hybridization. We also describe methods for the production of region-specific and chromosome-specific DNA probes and hybridization techniques for the visualization of these probes.

Animals↗

Induction of DNA adducts in vivo in rat lung cells by fume condensates of roofing asphalt.

Many workers in the highway construction and roofing industries are potentially exposed to asphalt fumes. However, little is known regarding the carcinogenic hazards of these fumes to the exposed workers. Previous studies have shown that condensates of asphalt fumes are weakly mutagenic to bacteria and are capable of inducing micronucleus formation in cultured mammalian cells. In this study, the induction of DNA adducts in vivo in lung and white blood cells (WBCs) of rats by fume condensates of type I and type III roofing asphalts was investigated using 32P-postlabeling analysis. Male CD rats (3/group) received 3 intratracheal instillations of fume condensates in a 24-h period. DNA from both lung cells and WBCs were isolated and used to detect DNA adducts. Condensates of both roofing asphalt fumes caused DNA adduct formation in rat lung cells in a similar dose-related manner. Under the conditions studied, however, neither type I nor type III fume condensate induced DNA adducts in WBCs. These results indicate that 1) condensates of fumes from both type I and type III have similar genotoxic activity, 2) chemicals in the condensates of roofing asphalt fumes can covalently bind to the DNA of rat lung cells, and 3) WBCs may not be a suitable surrogate for lung cells in DNA adduct studies of workers exposed to roofing asphalt fumes.

Animals↗

Chronic ingestion of clastogens by mice and the frequency of chromosome aberrations.

Environmental exposure to mutagens is believed to play a significant role in human carcinogenesis. Determination of the in vivo effects of a single mutagen is best done in laboratory animals because humans are exposed to a variety of mutagens both in their diet and in the rest of their environment. In this study, C57BL/6N female mice were used to analyze the effect on chromosomes of chronic ingestion of a mutagen dissolved in drinking water. Cyclophosphamide (CP) or urethane (ethyl carbamate, EC) were dissolved in sterile drinking water at concentrations of 0, 32, 64, and 96 ppm or 0, 5,000, 10,000, and 15,000 ppm, respectively. All exposures began at 8 weeks of age and continued through the 20th week unless terminated earlier due to toxicity. Body weights and water consumption were measured weekly. Blood and bone marrow were taken from approximately five mice per exposure group at 4, 8, and 12 weeks from the start of exposure. All mice remaining after 12 weeks received drinking water without any carcinogen for an additional 6 weeks to determine if induced aberrations persisted. Chromosome translocations, measured by painting, were not induced in blood or bone marrow cells at any time point for either chemical. However, both carcinogens induced significant increases in micronucleated normochromatic erythrocytes, indicating that the carcinogens reached the tissues examined in these experiments. These results indicate that chronic exposure of mice to chemical carcinogens induces chromosome breakage measurable by micronuclei. However, the breakage and reunion necessary to see chromosome exchanges such as translocations were not observed in this study.

Administration, Oral↗

Successful medical management of orbital abscess in a patient with sickle cell anemia.

The authors describe a patient with sickle cell anemia who had an orbital abscess at the site of a bone infarct during hospitalization for a painful crisis. Because the patient was in close medical observation, the orbital abscess was diagnosed within 48 hours of the onset of symptoms. The patient was treated with a 2-week course of intravenous antibiotics. This resulted in complete resolution of the abscess, as evidenced by clinical improvement and findings on computerized axial tomography scanning. The authors conclude that a heightened suspicion of orbital abscess in sickle cell patients with ocular symptoms will allow the diagnosis of an orbital abscess that can then be cured with antibiotic treatment but without orbital surgery.

Abscess↗

Preneoplastic potential of morphologically distinct transformed foci induced by 3-methylcholanthrene.

Individual variability of scoring foci positive for transformation presents a difficult problem in assessing the transformation assay. In this study, an attempt was made to identify five morphologically distinct types of transformed foci based on size (2-3, 3-4, and > or = 4 mm in diameter), invasiveness (smooth vs. invading margins), and other properties (piling vs. spread) induced by 3-methylcholanthrene in Balb/c-3T3 cells. The transformed focal cells were used in in vitro studies including anchorage-independent analysis, focal reconstruction, gene transfection using NIH-3T3 host cells, and Southern blotting to assess amplification of five proto-oncogenes (K-ras, H-ras, c-fos, c-jun, c-myc) and a tumor suppressor (p53) gene. Results showed that 1) there was a significant increase in anchorage-independent growth of all five types of foci ranging from 7-12%; 2) all five morphological types of transformed foci showed 8-15% focal reconstruction; 3) DNA from all five types of transformed foci induced transformation in NIH-3T3 cells at a level significantly above the control DNA; 4) gene amplification studies indicated amplification in both K-ras and H-ras proto-oncogenes; however, c-fos, c-jun, and c-myc did not show DNA amplification. The tumor suppressor gene (p53) was activated and the increase was up to 3-fold over the normal Balb/c-3T3 DNA. These findings are consistent with our hypothesis that all five morphologically different foci have preneoplastic potential and that any foci of size > or = 2 mm regardless of invasiveness and piling should be scored as positive during the transformation assay.

3T3 Cells↗

Modulation of human neutrophil inflammatory responses by nitric oxide: studies in unprimed and LPS-primed cells.

Because nitric oxide (NO) can act both as a regulatory and as a toxic molecule, we have studied N-formyl-methionyl-leucyl-phenylalanine (fMLF)-stimulated responses of human neutrophils (PMNs) during various conditions of NO modulation in unprimed and bacterial lipopolysaccharide (LPS) -primed cells. Effects of various NO modulators were assessed on stimulated superoxide (O2-) generation, granule exocytosis, homotypic aggregation, and rises in intracellular free Ca2+ ([Ca2+]i). Significant differences in the effects of various NO modulators on inflammatory responses of PMNs kept in stirred suspension versus those kept under static and/or adherent conditions, were observed. L-arginine, the physiological substrate for NO synthase (NOS), and NG-nitro-L-arginine methyl ester, an inhibitor of NOS, both caused a 40-50% inhibition of LPS-induced priming of O2- generation in PMNs in stirred suspension, but not in LPS-primed PMNs under static or adherent conditions. The NO donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, completely abrogated the LPS-induced priming of O2- generation in PMNs in suspension, while causing only a 40-50% inhibition in PMNs under static or adherent conditions. The Ca2+ ionophore, A23187, prevented the LPS-induced priming of O2- generation without affecting O2- generation in unprimed PMNs. LPS priming of PMNs induced about a twofold increase in fMLF-stimulated homotypic aggregation, exocytosis of secondary granules, and rises in [Ca2+]i. In related studies, we also provide definitive evidence for enzymatic formation of NO in human PMNs and demonstrate a significant decrease in NO levels in LPS-primed PMNs. Taken together, these findings indicate that NO modulates PMN inflammatory responses and plays a protective role in priming and activation processes of inflammatory PMNs.

Humans↗

Colchicine effects on meiosis in the male mouse. I. Meiotic prophase: synaptic arrest, univalents, loss of damaged spermatocytes and a possible checkpoint at pachytene.

Antimitotic agents administered at the time of synapsis (leptotene/zygotene) have been shown to induce synaptic abnormalities visible during pachytene in the male mouse. The object of this study was to test the hypothesis that cells with relatively large amounts of colchicine-induced damage to the synaptonemal complex (SC) are eliminated from prophase whereas cells with relatively small amounts of SC damage proceed through to the end of prophase. Male mice were injected with tritiated thymidine to mark a cohort of spermatocytes at premeiotic S-phase for tracking through pachytene. Forty-eight hours later, when those cells were at leptotene/zygotene, colchicine was administered intratesticularly. Whole-mount SC spreads were made from animals sacrificed at various times following colchicine administration, and prepared for autoradiography. The marked cells were examined by light and electron microscopy and the kind and number of synaptic abnormalities were scored throughout pachytene. Colchicine-induced SC damage included single axial elements (univalents), together with partially synapsed and nonhomologously synapsed SCs. The amount of SC damage (amount and type per cell and frequency of cells with damage) scored at early pachytene exceeded by three- to fivefold the amount at late pachytene. This is consistent with spermatogenic cell loss from the seminiferous tubule via colchicine-induced destruction of Sertoli cell microtubules. The presence of spermatocytes with no more than four autosomal univalents at late pachytene indicates that some cells with low amounts of synaptic damage progress to the end of pachytene. The loss of the most severely damaged cells may represent a meiotic checkpoint at early pachytene in the male mouse.

Animals↗

X chromosome inactivation and micronuclei in normal and Turner individuals.

Studies on aneuploidy have shown that the X is the most frequently lost chromosome in females, and that the number of X chromosome-positive micronuclei increases with age in women. Recently, we showed that the inactive X chromosome is incorporated preferentially in micronuclei. The objectives of the current study were, firstly, to determine the incidence of X chromosome incorporation into micronuclei in males and, secondly, to determine the incidence of X chromosome incorporation into micronuclei of females with Turner syndrome. Blood samples were obtained from 18 male newborns and 35 normal adult males ranging in age from 22 to 79 years and from seven women with non-mosaic Turner syndrome aged 11-39 years. Isolated lymphocytes were cultured in the presence of cytochalasin B and 2000 binucleated cells per subject were scored for micronuclei. Cells were then hybridized with the biotinylated X centromere-specific probe, pBamX7, and visualized with fluorescein-conjugated avidin. All micronucleated cells were relocated and evaluated for the presence or absence of the X chromosome. Of the 335 micronuclei observed, 6.6% (22/335) contained an X chromosome. Analysis of variance shows a statistically significant increase, for both males and Turner females, in the number of X chromosome-positive micronuclei with age (P < 0.001). These data also show that the X chromosome is included in micronuclei from males more often than would be expected by chance (P < 0.005; chi 2 analysis, 15 df). Here we show that there is a tenfold difference in the frequency of X chromosome-positive micronuclei in 46,XX females compared to 46,XY males and 45,X females, providing further support to our previous finding that the X chromosome in micronuclei is the inactive chromosome.

Adolescent↗

The persistence of aberrations in mice induced by gamma radiation as measured by chromosome painting.

Fluorescence in situ hybridization, or chromosome painting, has become an invaluable tool in the cytogenetic evaluation of historical or chronic exposure because it can be used to detect stable genetic damage, such as translocations, which persist through cell division, quickly and easily. The recent development of chromosome-specific composite DNA probes for the mouse has allowed the use of chromosome painting in this commonly used animal model. In order to measure the persistence of radiation-induced translocations, C57BL/6 female mice were given a whole body acute dose of 0, 1, 2, 3 or 4 Gy 137Cs gamma rays at 8 weeks of age. Metaphase chromosomes from both peripheral blood and bone marrow cells were obtained from four mice in each dose group at 1, 8, 15 and 30 days post-irradiation. Chromosomes 2 and 8 were painted, while the remaining chromosomes were counterstained with propidium iodide. DAPI counterstain was used to differentiate between translocations and dicentrics because it brightly labels the centromeric heterochromatin. The equivalent of 100 cells from each tissue was scored from each mouse. The results show that the percentage of reciprocal translocations, at least at doses of 3 Gy or lower, did not decrease with time in either tissue. In contrast, the frequency of non-reciprocal translocations induced by doses of 3 Gy or lower, remained unchanged in the peripheral blood, but decreased after a week in the bone marrow, then remained constant. An increase in these two types of aberration was observed between 15 and 30 days in the bone marrow and may have been due to clonal expansion. Dicentrics decreased with time in both tissues, almost none remained in the bone marrow after 8 days. These data suggest that reciprocal translocations are persistent and will serve as an effective biodosimeter for radiation exposure.

Animals↗

In vitro studies on the genotoxicity of 2,4-dichloro-6-nitrophenol ammonium (DCNPA) and its major metabolite.

2,4-dichloro-6-nitrophenol ammonium (DCNPA) is used as a herbicide. However, information on the potential health hazards of DCNPA is limited. In a previous study, we found that DCNPA is genotoxic to Bacillus subtilis and yeast. Further studies were performed to determine whether DCNPA and its major metabolite, 2,4-dichloro-6-aminophenol (DCAP), can induce reverse mutations in Salmonella, gene mutations at the HPRT locus, sister chromatid exchanges (SCEs) and micronuclei (MN) in V79 cells. Results show that DCNPA does not produce a positive response for any endpoint at concentrations tested. However, treatment of V79 cultures with DCAP caused a significant increase in SCEs and MN in a concentration-dependent manner. These results indicate that DCAP damages DNA and causes chromosomal aberrations in V79 cells. Therefore, DCNPA could pose potential health hazards to populations exposed to this herbicide.

Aminophenols↗