Risk of bladder cancer in male Japanese workers exposed to ortho-toluidine and other aromatic amines.

PURPOSE: Nine bladder cancer (BCa) cases were reported among aromatic amine-exposed male workers at a factory manufacturing organic dye/pigment intermediates in Japan. We aimed to evaluate the characteristics of aromatic amine-exposed workers by cross-sectional observation, and the risk of BCa by assessing the standardized incidence ratio (SIR). METHODS: In the cross-sectional study, our subjects were: 9 BCa patients, 36 aromatic amine-exposed non-patients, and 79 non-exposed workers from 3 factories. We evaluated the subjects' medical history, urinalysis, qualitative determination of nuclear matrix protein 22, and urinary cytology. For SIR assessment, 98 aromatic amine-exposed workers from 1 factory were included, and the Japanese general male population was used as a referent population. Since no direct aromatic amine-exposure data were available, we calculated surrogate exposure levels using information on job sites, exposure potency, and duration. RESULTS: Coexistent aromatic amines were ortho-toluidine (OT), aniline, para-toluidine, ortho-anisidine, 2,4-xylidine, and ortho-chloroaniline. The prevalence rates of cystitis and bladder lesion-related symptoms in both BCa patients and aromatic amine-exposed non-patient workers were significantly higher than those of non-exposed workers. Overall, the SIR for BCa in OT-exposed workers was 56.8 (95% CI 27.7-104.3) and apparent dose-response relationships were revealed between the SIR and the surrogate exposure level in the 0-10-year lagged analyses. Overall, SIRs in other aromatic amine-exposed workers were also significantly high but no or unclear dose-response relationships were observed. CONCLUSIONS: We conclude that OT may be responsible for the increased risk of BCa. Regular monitoring of bladder lesion-related symptoms is essential for the early identification of BCa.

A weight of evidence assessment of the genotoxicity of 2,6-xylidine based on existing and new data, with relevance to safety of lidocaine exposure.

Lidocaine has not been associated with cancer in humans despite 8 decades of therapeutic use. Its metabolite, 2,6-xylidine, is a rat carcinogen, believed to induce genotoxicity via N-hydroxylation and DNA adduct formation, a non-threshold mechanism of action. To better understand this dichotomy, we review literature pertaining to metabolic activation and genotoxicity of 2,6-xylidine, identifying that it appears resistant to N-hydroxylation and instead metabolises almost exclusively to DMAP (an aminophenol). At high exposures (sufficient to saturate phase 2 metabolism), this may undergo metabolic threshold-dependent activation to a quinone-imine with potential to redox cycle producing ROS, inducing cytotoxicity and genotoxicity. A new rat study found no evidence of genotoxicity in vivo based on micronuclei in bone marrow, comets in nasal tissue or female liver, despite high level exposure to 2,6-xylidine (including metabolites). In male liver, weak dose-related comet increases, within the historical control range, were associated with metabolic overload and acute systemic toxicity. Benchmark dose analysis confirmed a non-linear dose response. The weight of evidence indicates 2,6-xylidine is a non-direct acting (metabolic threshold-dependent) genotoxin, and is not genotoxic in vivo in rats in the absence of acute systemic toxic effects, which occur at levels 35 × beyond lidocaine-related exposure in humans.

More methemoglobin is produced by benzocaine treatment than lidocaine treatment in human in vitro systems.

The clinical use of local anesthetic products to anesthetize mucous membranes has been associated with methemoglobinemia (MetHba), a serious condition in which the blood has reduced capacity to carry oxygen. An evaluation of spontaneous adverse event reporting of MetHba submitted to FDA through 2013 identified 375 reports associated with benzocaine and 16 reports associated with lidocaine. The current study was performed to determine the relative ability of benzocaine and lidocaine to produce methemoglobin (MetHb) in vitro. Incubation of 500µM benzocaine with whole human blood and pooled human liver S9 over 5h resulted in MetHb levels equaling 39.8±1.2% of the total hemoglobin. No MetHb formation was detected for 500µM lidocaine under the same conditions. Because liver S9 does not readily form lidocaine hydrolytic metabolites based on xylidine, a primary metabolic pathway, 500µM xylidine was directly incubated with whole blood and S9. Under these conditions MetHb levels of 4.4±0.4% were reached by 5h. Studies with recombinant cytochrome P450 revealed benzocaine to be extensively metabolized by CYP 1A2, with 2B6, 2C19, 2D6, and 2E1 also having activity. We conclude that benzocaine produces much more MetHb in in vitro systems than lidocaine or xylidine and that benzocaine should be more likely to cause MetHba in vivo as well.

An innovative nanoparticle-modified carbon paste sensor for ultrasensitive detection of lignocaine and its extremely carcinogenic metabolite residues in bovine food samples: Application of NEMI, ESA, AGREE, ComplexGAPI, and RGB12 algorithms.

Nowadays, veterinary medicine residues have been viewed as a major threat to food safety worldwide, especially when dealing with carcinogenic residues. Herein, we present the first differential pulse voltammetric method for the quantification of lignocaine and its carcinogenic metabolite 2,6-xylidine residues in bovine food samples, aided by five greenness and whiteness assessment tools, including NEMI, ESA, ComplexGAPI, AGREE, and RGB12. The method depends on the electrochemical oxidation after modification of the carbon paste sensor with recycled Al2O3-NPs functionalized multi-walled carbon nanoparticles. The produced sensor (Al2O3-NPs/MWCNTs/CPE) was characterized using XRD, FT-IR, EDX, SEM, and TEM. As expected, the active surface area and electron transfer processes were accelerated by the modification, resulting in ultra-sensitive quantification with detection limits of 19.00 and 13.94 nM for lignocaine and 2,6-xylidine, respectively. In terms of greenness, whiteness, sustainability, analytical effectiveness, and economic and practical considerations, the proposed method outperforms the reported methods.

Effect of 2,6-xylidine (DMA) on secretion of biomarkers for inflammation and neurodevelopment by the placenta.

Cigarette smoke enhances placental inflammation and interferes with steroidogenesis. However, the chemicals in the smoke responsible for these biological activities are unclear. 2,6 xylidine (also called 2,6 Dimethylaniline, DMA) is a component of cigarette smoke that has carcinogenic properties but its effects on the placenta are unknown. Therefore, we hypothesized that DMA may interfere with placental steroidogenesis or enhance placental inflammation. Placental explant cultures were treated with 0-50,000 nM DMA and concentrations of progesterone (P4), estradiol (E2), testosterone (T), IL-1ß, TNF-α, IL-6, sgp130, HO-1, IL-10, 8-Isoprostane (8-IsoP), and BDNF in the conditioned medium were quantified. Since many environmental toxins enhance the proinflammatory host response to infection, we also performed experiments on placental cultures co-stimulated with 107 heat-killed E. coli. DMA alone significantly reduced P4 and T secretion but enhanced E2 secretion. The toxin also reduced placental secretion of IL-6, sgp130, and BDNF. For bacteria-stimulated cultures, DMA increased secretion of P4 and T, and proinflammatory cytokines (IL-1ß, TNF-α) but had mixed effects on anti-inflammatory markers, increasing some (sgp130, IL-10) and reducing others (HO-1). However, DMA enhanced 8-IsoP levels by bacteria-stimulated placental cultures, suggesting that it increases oxidative stress by the tissues. These studies suggest that DMA affects secretion of biomarkers by the placenta and may promote inflammation. Further studies are needed to determine if these observed changes occur in vivo and the extent to which DMA exposure increases the risk of adverse pregnancy outcomes associated with smoking in pregnancy.

Histology of Damage Caused by Euschistus heros (F.) Nymphs in Soybean Pods and Seeds.

The Neotropical brown stink bug, Euschistus heros (F.), feeds on stems, leaves, pods, and seeds of soybean, Glycine max (L.) Merrill. Knowledge of the damage that nymphs at different instars can cause to soybean pods and seeds, as well as efficient histological techniques for locating the salivary sheath are sparse. This study developed a new double-staining method to facilitate distinguishing the salivary sheath from plant tissues and to anatomically evaluate the damage caused by nymphs of different instars as they feed on soybean pods and seeds. Five insects from each of the analyzed instars (1st, 2nd, 3rd, 4th, and 5th) per pod at the R6 stage (full pod-filling) were kept in clip cages for 48 h of feeding. The salivary sheath was analyzed to localize the damage (pod, vascular bundle, and seed) and the depth reached by the damage (categorized tissue). Double staining with xylidine ponceau and toluidine blue provided the best differentiation between the salivary sheath and watery sheath (proteins stained red) and the plant tissues (stained blue). First instar nymphs do not feed. Second instar and older nymphs caused damage to seeds, which became more severe with later developmental stages. The damage consists of coalescence of protein bodies and degradation and breakdown of the cell wall, marked by darkened regions in the embryo tissue of seeds. The information generated will contribute to new studies on feeding habits and emphasizes the need to control E. heros in early development stages.

Production of laccase by Pynoporus sanguineus using 2,5 - Xylidine and ethanol.

Enzyme application in biotechnological and environmental processes has had increasing interest due to its efficiency, selectivity and mainly for being environmentally healthful, but these applications require a great volume of enzymes. In this work the effect of different concentrations of ethanol and 2,5-xylidine on growth and production of laccase by Pycnoporus sanguineus was investigated. In a medium containing 200 mg.L(-1) of 2,5-xylidine or 50 g.L(-1) of ethanol, the maximum activity of laccase was 2019 U.L(-1) and 1035 U.L(-1), respectively. No direct correlation between biomass and activity of laccase was observed for any of the inducers used during the tests. Ethanol concentrations, larger than or equal to 20 g.L(-1), inhibited the radial growth of P. sanguineus. This study showed that ethanol, which has less toxicity and cost than the majority of the studied inducers, presents promising perspectives for laccase production by P. sanguineus.

Disulfonated azo dyes: metal coordination and ion-pair separation in twelve MII compounds of Ponceau Xylidine and Crystal Scarlet.

The structures of seven divalent metal cation compounds of Ponceau Xylidine {PX; systematic name of dication: 4-[2-(3,4-dimethylphenyl)hydrazin-1-ylidene]-3-oxo-3,4-dihydronaphthalene-2,7-disulfonate}, also known as Acid Red 26, CI 16150, and of five divalent metal cation compounds of Crystal Scarlet {CS; systematic name of dication: 8-[2-(naphthalen-1-yl)hydrazin-1-ylidene]-7-oxo-7,8-dihydronaphthalene-1,3-disulfonate}, also known as Acid Red 44, CI 16250, are presented. These are hexaaquamagnesium(II) PX dimethylformamide (DMF) monosolvate, [Mg(H2O)6](C18H14N2O7S2)·C3H7NO, (I); heptaaquacalcium(II) PX 2.5-hydrate, [Ca(H2O)7](C18H14N2O7S2)·2.5H2O, (II); catena-poly[aqua(µ-DMF)tris(DMF)bis(µ3-PX)distrontium(II)], [Sr(C18H14N2O7S2)(C3H7NO)2(H2O)0.5]n, (III); the transition-metal series hexaaquametal(II) PX DMF monosolvate, [M(H2O)6](C18H14N2O7S2)·C3H7NO, where M (metal) = Co, (IV), Ni, (V), Cu, (VI), and Zn, (VII); heptaaquacalcium(II) CS monohydrate, [Ca(H2O)7](C20H13N2O7S2)·H2O, (VIII); octaaquastrontium(II) CS monohydrate, [Sr(H2O)8](C20H13N2O7S2)·H2O, (IX); catena-poly[[triaqua(DMF)barium(II)]-µ-CS], [Ba(C20H13N2O7S2)(C3H7NO)(H2O)3]n, (X); tetrakis(DMF)(CS)copper(II) monohydrate, [Cu(C20H13N2O7S2)(C3H7NO)4]·H2O, (XI); and catena-poly[[[aquatris(DMF)zinc(III)]-µ-CS] diethyl ether hemisolvate], {[Zn(C20H13N2O7S2)(C3H7NO)3(H2O)]·0.5C4H10O}n, (XII). In all cases, the structures obtained were solvates with dimethylformamide (DMF) and/or water present. The disulfonated naphthalene-based azo anions adopt hydrazone tautomeric forms. The structures of the Mg salt and of four transition-metal forms (M = Co, Ni, Cu and Zn) of PX are found to form an isostructural series. All have solvent-separated ion-pair (SSIP) type structures and the formula [M(H2O)6][PX]·DMF. The Ca salt of PX also has an SSIP structure, but has a higher hydration state, [Ca(H2O)7][PX]·2.5H2O. In contrast, the Sr salt of PX, [Sr(PX)(DMF)2(H2O)0.5]n forms a one-dimensional coordination polymer. Both the Ca and the Sr salt of CS have an SSIP structure, namely [Ca(H2O)7][CS]·H2O and [Sr(H2O)8][CS]·H2O, whilst the heavier Ba analogue, [Ba(CS)(DMF)(H2O)3]n, forms a one-dimensional coordination polymer. Unlike PX, two CS structures containing transition metals are found to be coordination complexes, [Cu(CS)(DMF)4]·H2O and {[Zn(CS)(DMF)3(H2O)]·0.5Et2O}n. This suggests that CS is a better ligand than PX for transition metals. The Cu complex forms discrete molecules with Cu in a square-pyramidal environment, whilst the Zn species is a one-dimensional coordination polymer based on octahedral Zn centres.

An epidemic of bladder cancer: ten cases of bladder cancer in male Japanese workers exposed to ortho-toluidine.

BACKGROUND: ortho-Toluidine (OT) was listed as a Group 1 carcinogen by the International Agency for Research on Cancer in 2012 based on epidemiological observations of workers co-exposed to OT and aromatic amines. From 2014 to 2017, several cases of bladder cancer (BCa) secondary to occupational exposure, primarily to OT, were detected in Japan. OBJECTIVE: To describe 10 cases of BCa in male Japanese workers exposed primarily to OT at two plants that produce organic dye and pigment intermediates. METHODS: Details of the 10 cases were obtained from company records and through a questionnaire and interview. The surrogate level of exposure to each aromatic amine was calculated as the total job-weight/month for each process for each job-year. RESULTS: No quantitative exposure data were available. In most cases the surrogate level of exposure to OT was higher than to other amines. All 10 cases were exposed primarily to OT and co-exposed to para-toluidine, ortho-anisidine, aniline, 2,4-xylidine or ortho-chloroaniline. The age range at diagnosis was 41-71 years (mean 56). The duration of OT exposure was 7-28 years (mean 16.5). Disease latency was 16-28 years (mean 21.9). Eight patients were smokers. The main symptom at diagnosis was hematuria (70%). CONCLUSIONS: The characteristics of BCa cases were associated with a high surrogate level of OT exposure and a disease latency of more than 20 years from the initial OT exposure. The main route of OT exposure was likely through the skin. It is necessary to continue health examinations in these target groups.

Measurement of lidocaine and 2,6-dimethylaniline in minipig plasma, skin, and dermal tapes using UHPLC with electrospray MS/MS.

Sensitive LC-MS/MS methods were developed to measure lidocaine and its metabolite 2,6-dimethylaniline (2,6-DMA) with application to transdermal studies. The methods for lidocaine in minipig plasma, tissue biopsies, and dermal tapes utilized mixed mode/SCX solid phase extraction, with lower quantitation limits of 25 pg/mL in plasma, 15 ng/g tissue, and 5 ng/tape. 2,6-DMA was measured in plasma and skin tissue homogenates by ultrafiltration and (for tissue) by further derivatization with 4-methoxybenzoyl chloride to form the corresponding benzamide derivative, which extended the lower limit of quantitation to 200 pg/mL. The methods allowed local measurement of lidocaine in stratum corneum, punch biopsies, and plasma and of 2,6-DMA in plasma and biopsies obtained from minipigs dosed with experimental transdermal formulations. Quantitation limits were approximately 7-fold lower than previously reported for lidocaine and 3-fold lower for 2,6-DMA.

Interaction between lidocaine hydrochloride (with and without adrenaline) and various irrigants: A nuclear magnetic resonance analysis.

BACKGROUND: Interaction between local anesthetic solution, lidocaine hydrochloride (with and without adrenaline), and root canal irrigants such as sodium hypochlorite (NaOCl), ethylene diamine tetra-acetic acid (EDTA), and chlorhexidine (CHX) has not been studied earlier. Hence, the purpose of this in vitro study was to evaluate the chemical interaction between 2% lidocaine hydrochloride (with and without adrenaline) and commonly used root canal irrigants, NaOCl, EDTA, and CHX. MATERIALS AND METHODS: SAMPLES WERE DIVIDED INTO EIGHT EXPERIMENTAL GROUPS: Group I-Lidocaine hydrochloride (with adrenaline)/3% NaOCl, Group II-Lidocaine hydrochloride (with adrenaline)/17% EDTA, Group III- Lidocaine hydrochloride (with adrenaline)/2% CHX, Group IV-Lidocaine hydrochloride (without adrenaline)/3% NaOCl, Group V-Lidocaine hydrochloride (without adrenaline)/17% EDTA, Group VI-Lidocaine hydrochloride (without adrenaline)/2% CHX, and two control groups: Group VII-Lidocaine hydrochloride (with adrenaline)/deionized water and Group VIII-Lidocaine hydrochloride (without adrenaline)/deionized water. The respective solutions of various groups were mixed in equal proportions (1 ml each) and observed for precipitate formation. Chemical composition of the formed precipitate was then analysed by nuclear magnetic resonance spectroscopy (NMR) and confirmed with diazotation test. RESULTS: In groups I and IV, a white precipitate was observed in all the samples on mixing the respective solutions, which showed a color change to reddish brown after 15 minutes. This precipitate was then analysed by NMR spectroscopy and was observed to be 2,6-xylidine, a reported toxic compound. The experimental groups II, III, V, and VI and control groups VII and VIII showed no precipitate formation in any of the respective samples, until 2 hours. CONCLUSION: Interaction between lidocaine hydrochloride (with and without adrenaline) and NaOCl showed precipitate formation containing 2,6-xylidine, a toxic compound.

Production of laccase by Pynoporus sanguineus using 2, 5 - Xylidine and ethanol

Enzyme application in biotechnological and environmental processes has had increasing interest due to its efficiency, selectivity and mainly for being environmentally healthful, but these applications require a great volume of enzymes. In this work the effect of different concentrations of ethanol and 2,5 - xylidine on growth and production of laccase by Pycnoporus sanguineus was investigated. In a medium containing 200 mg.L-1 of 2,5 - xylidine or 50 g.L-1 of ethanol, the maximum activity of laccase was 2019 U.L-1 and 1035 U.L-1, respectively. No direct correlation between biomass and activity of laccase was observed for any of the inducers used during the tests. Ethanol concentrations, larger than or equal to 20 g.L-1, inhibited the radial growth of P. sanguineus. This study showed that ethanol, which has less toxicity and cost than the majority of the studied inducers, presents promising perspectives for laccase production by P. sanguineus.