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Background and Purpose: Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disease belonging to the inherited bone marrow failure syndromes and characterized by hypocellular bone marrow, exocrine pancreatic insufficiency, and skeletal abnormalities. SDS is associated with increased risk of developing myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). Although SDS is not primarily considered an inflammatory disorder, some of the associated conditions (e.g., neutropenia, pancreatitis and bone marrow dysfunction) may involve inflammation or immune system dysfunctions. We have already demonstrated that signal transducer and activator of transcription (STAT)-3 and mammalian target of rapamycin (mTOR) were hyperactivated and associated with elevated IL-6 levels in SDS leukocytes. In this study, we analyzed the level of phosphoproteins involved in STAT3 and mTOR pathways in SDS lymphoblastoid cells (LCLs) and the secretomic profile of soluble pro-inflammatory mediators in SDS plasma and LCLs in order to investigate the systemic inflammation in these patients and relative pathways., Methods: Twenty-six SDS patients and seven healthy donors of comparable age were recruited during the programmed follow-up visits for clinical evaluation at the Verona Cystic Fibrosis Center Human. The obtained samples (plasma and/or LCLs) were analyzed for: phosphoproteins, cytokines, chemokines and growth factors levels by Bio-plex technology; microRNAs profiling by next generation sequencing (NGS) and microRNAs expression validation by Real Time-PCR (RT-PCR) and droplet digital PCR (ddPCR) ., Results: We demonstrated dysregulation of ERK1/2 and AKT phosphoproteins in SDS, as their involvement in the hyperactivation of the STAT3 and mTOR pathways confirmed the interplay of these pathways in SDS pathophysiology. However, both these signaling pathways are strongly influenced by the inflammatory environment. Here, we reported that SDS is characterized by elevated plasma levels of several soluble proinflammatory mediators. In vitro experiments show that these pro-inflammatory genes are closely correlated with STAT3/mTOR pathway activation. In addition, we found that miR-181a-3p is down-regulated in SDS. Since this miRNA acts as a regulator of several pro-inflammatory pathways such as STAT3 and ERK1/2, its down-regulation may be a driver of the constitutive inflammation observed in SDS patients., Conclusions: The results obtained in this study shed light on the complex pathogenetic mechanism underlying bone marrow failure and leukemogenesis in SDS, suggesting the need for anti-inflammatory therapies for SDS patients., Competing Interests: Declarations. Ethics approval and consent to participate: This study was approved by the Ethics Committee of Azienda Ospedaliera Universitaria Integrata (Verona, Italy: approval No. 4182 CESC). Informed consent was obtained from participants or their legal representatives. Consent for publication: All the authors agree to publish. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Humans are potentially exposed to a large amount of chemicals present in the environment and in the workplace. In the European Human Biomonitoring initiative (Human Biomonitoring for the European Union = HBM4EU), acrylamide, mycotoxins (aflatoxin B1, deoxynivalenol, fumonisin B1), diisocyanates (4,4'-methylenediphenyl diisocyanate, 2,4- and 2,6-toluene diisocyanate), and pyrethroids were included among the prioritized chemicals of concern for human health. For the present literature review, the analytical methods used in worldwide biomonitoring studies for these compounds were collected and presented in comprehensive tables, including the following parameter: determined biomarker, matrix, sample amount, work-up procedure, available laboratory quality assurance and quality assessment information, analytical techniques, and limit of detection. Based on the data presented in these tables, the most suitable methods were recommended. According to the paradigm of biomonitoring, the information about two different biomarkers of exposure was evaluated: a) internal dose = parent compounds and metabolites in urine and blood; and b) the biologically effective = dose measured as blood protein adducts. Urine was the preferred matrix used for deoxynivalenol, fumonisin B1, and pyrethroids (biomarkers of internal dose). Markers of the biological effective dose were determined as hemoglobin adducts for diisocyanates and acrylamide, and as serum-albumin-adducts of aflatoxin B1 and diisocyanates. The analyses and quantitation of the protein adducts in blood or the metabolites in urine were mostly performed with LC-MS/MS or GC-MS in the presence of isotope-labeled internal standards. This review also addresses the critical aspects of the application, use and selection of biomarkers. For future biomonitoring studies, a more comprehensive approach is discussed to broaden the selection of compounds., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The enzyme that, in Saccharomyces cerevisiae, catalyzes the last step in both proline synthesis and arginine catabolism, δ 1 -pyrroline-5-carboxylate (P5C) reductase, was purified to near homogeneity and characterized thoroughly. Retention patterns upon gel permeation chromatography were consistent with a homodecameric composition of the holomer. High lability of the purified preparations and stabilization by reducing compounds suggested susceptibility to reactive-oxygen-species-mediated damage. Both NADH and NADPH were used as the electron donor, the latter resulting in a 3-fold higher V max . However, a higher affinity toward NADH was evident, and the NADPH-dependent activity was inhibited by NAD + , proline, arginine, and a variety of anions. With proline and arginine, the inhibition was of the competitive type with respect to the specific substrate, and of the uncompetitive- or mixed-type with respect to NADPH, respectively. The results suggest that, contrary to the enzyme from higher plants, yeast P5C reductase may preferentially use NADH in vivo. An in silico analysis was also performed to investigate the structural basis of such enzyme features. Superposition of the protein model with the experimental structure of P5C reductase from Medicago truncatula allowed us to hypothesize on the possible allosteric sites for arginine and anion binding, and the cysteine pairs that may be involved in disulfide formation.

High levels of some enzymes involved in proline synthesis and utilization were early found in soybean nodules, and rhizobial knockout mutants were shown to be defective in inducing nodulation and/or fixing nitrogen, leading to postulate that this amino acid may represent a main substrate for energy transfer from the plant to the symbiont. However, inconsistent results were reported in other species, and several studies suggested that proline metabolism may play an essential role in the legume- Rhizobium symbiosis only under stress. Different mechanisms have been hypothesized to explain the beneficial effects of proline on nodule formation and bacteroid differentiation, yet none of them has been conclusively proven. Here, we summarize these findings, with special emphasis on the occurrence of a legume-specific isoform of δ 1 -pyrroline-5-carboxylate synthetase, the enzyme that catalyses the rate-limiting step in proline synthesis. Data are discussed in view of recent results connecting the regulation of both, the onset of nodulation and proline metabolism, to the redox status of the cell. Full comprehension of these aspects could open new perspectives to improve the adaptation of legumes to environmental stress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sabbioni and Forlani.)

In certain cancers, such as breast, prostate and some lung and skin cancers, the gene for the enzyme catalysing the second and last step in proline synthesis, δ 1 -pyrroline-5-carboxylate (P5C) reductase, has been found upregulated. This leads to a higher proline content that exacerbates the effects of the so-called proline-P5C cycle, with tumour cells effectively using this method to increase cell survival. If a method of reducing or inhibiting P5C reductase could be discovered, it would provide new means of treating cancer. To address this point, the effect of some phenyl-substituted derivatives of aminomethylene-bisphosphonic acid, previously found to interfere with the catalytic activity of plant and bacterial P5C reductases, was evaluated in vitro on the human isoform 1 (PYCR1), expressed in E. coli and affinity purified. The 3.5-dibromophenyl- and 3.5-dichlorophenyl-derivatives showed a remarkable effectiveness, with IC 50 values lower than 1 µM and a mechanism of competitive type against both P5C and NADPH. The actual occurrence in vivo of enzyme inhibition was assessed on myelogenous erythroleukemic K562 and epithelial breast cancer MDA-MB-231 cell lines, whose growth was progressively impaired by concentrations of the dibromo derivative ranging from 10 -6 to 10 -4  M. Interestingly, growth inhibition was not relieved by the exogenous supply of proline, suggesting that the effect relies on the interference with the proline-P5C cycle, and not on proline starvation.

Under several stress conditions, such as excess salt and drought, many plants accumulate proline inside the cell, which is believed to help counteracting the adverse effects of low water potential. This increase mainly relies upon transcriptional induction of δ 1 -pyrroline-5-carboxylate synthetase (P5CS), the enzyme that catalyzes the first two steps in proline biosynthesis from glutamate. P5CS mediates both the phosphorylation of glutamate and the reduction of γ-glutamylphosphate to glutamate-5-semialdehyde, which spontaneously cyclizes to δ 1 -pyrroline-5-carboxylate (P5C). In most higher plants, two isoforms of P5CS have been found, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate the regulation of P5CS at the transcriptional level, to date, the properties of the enzyme have been only poorly studied. As a consequence, the descriptions of post-translational regulatory mechanisms have largely been limited to feedback-inhibition by proline. Here, we report cloning and heterologous expression of P5CS2 from Oryza sativa . The protein has been fully characterized from a functional point of view, using an assay method that allows following the physiological reaction of the enzyme. Kinetic analyses show that the activity is subjected to a wide array of regulatory mechanisms, ranging from product inhibition to feedback inhibition by proline and other amino acids. These findings confirm long-hypothesized influences of both, the redox status of the cell and nitrogen availability, on proline biosynthesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sabbioni, Funck and Forlani.)

The major purpose of human biomonitoring is the mapping and assessment of human exposure to chemicals. The European initiative HBM4EU has prioritized seven substance groups and two metals relevant for human exposure: Phthalates and substitutes (1,2-cyclohexane dicarboxylic acid diisononyl ester, DINCH), bisphenols, per- and polyfluoroalkyl substances (PFASs), halogenated and organophosphorous flame retardants (HFRs and OPFRs), polycyclic aromatic hydrocarbons (PAHs), arylamines, cadmium and chromium. As a first step towards comparable European-wide data, the most suitable biomarkers, human matrices and analytical methods for each substance group or metal were selected from the scientific literature, based on a set of selection criteria. The biomarkers included parent compounds of PFASs and HFRs in serum, of bisphenols and arylamines in urine, metabolites of phthalates, DINCH, OPFRs and PAHs in urine as well as metals in blood and urine, with a preference to measure Cr in erythrocytes representing Cr (VI) exposure. High performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was the method of choice for bisphenols, PFASs, the HFR hexabromocyclododecane (HBCDD), phenolic HFRs as well as the metabolites of phthalates, DINCH, OPFRs and PAHs in urine. Gas chromatographic (GC) methods were selected for the remaining compounds, e.g. GC-low resolution MS with electron capture negative ionization (ECNI) for HFRs. Both GC-MS and LC-MS/MS were suitable for arylamines. New developments towards increased applications of GC-MS/MS may offer alternatives to GC-MS or LC-MS/MS approaches, e.g. for bisphenols. The metals were best determined by inductively coupled plasma (ICP)-MS, with the particular challenge of avoiding interferences in the Cd determination in urine. The evaluation process revealed research needs towards higher sensitivity and non-invasive sampling as well as a need for more stringent quality assurance/quality control applications and assessments., (Copyright © 2020. Published by Elsevier Ltd.)

Peptide nucleic acids (PNAs) are very useful tools for gene regulation at different levels, but in particular in the last years their use for targeting microRNA (anti-miR PNAs) has provided impressive advancements. In this respect, microRNAs related to the repression of cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis, are of great importance in the development of new type of treatments. In this paper we propose the use of an anti-miR PNA for targeting miR-145, a microRNA reported to suppress CFTR expression. Octaarginine-anti-miR PNA conjugates were delivered to Calu-3 cells, exerting sequence dependent targeting of miR-145-5p. This allowed to enhance expression of the miR-145 regulated CFTR gene, analyzed at mRNA (RT-qPCR, Reverse Transcription quantitative Polymerase Chain Reaction) and CFTR protein (Western blotting) level., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Objectives: 1,5-Naphthalene diisocyanate (NDI) is used in the plastic industry as a curing agent. 1,5-Naphthalene diisocyanate is classified as a sensitizing agent. The objective of this study has been to develop biomonitoring methods for the evaluation of exposure to NDI., Material and Methods: We obtained blood and urine samples from a group of 20 male workers exposed to NDI. The workers answered a questionnaire about their exposure history, job description, the number of years with the company and the time spent working with NDI over the 10 days of the study. Total plasma, albumin, and urine were analyzed for the presence of 1,5-naphthalenediamine (NDA) after acid hydrolysis using gas chromatography-mass spectrometry (GC-MS)., Results: 1,5-Naphthalenediamine was found in about 60% of the samples obtained from the workers. 1,5-Naphthalenediamine was obtained after acid hydrolysis of plasma, albumin, and urine at levels up to 1.5 pmol NDA/mg of plasma proteins, 1.15 pmol NDA/mg of albumin, and 55.3 pmol NDA/ml of urine, respectively., Conclusions: 1,5-Naphthalenediamine found in urine correlates best with the plasma levels (r = 0.91, p < 0.01). The albumin-adduct levels did not correlate with the NDI-specific immunoglobulin E (IgE) or total IgE present in the workers. The adduct and metabolite levels correlate with the air levels of NDI. Int J Occup Med Environ Health 2017;30(4):579-591., (This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.)

Purpose: The use of traditional cutting guides during knee arthroplasty in some cases could be extremely difficult, if not impossible, because of angular deformities, IM sclerosis, long-stemmed hip implants, or hardware within the femoral canal that cannot be removed. In these difficult cases navigation-assisted knee arthroplasty should be considered as an effective and appealing option., Methods: We present 14 cases in which ideal mechanical and prosthetic alignment was achieved with different image-free, computer-assisted navigation systems, because of an extra-articular deformity (group A, nine patients) or because of a retained implant or hardware (group B, five patients)., Results: After a mean follow-up of 28 months (range 12-53 months), the average knee score increased overall from a mean of 33 points (range 12-63) to 78 points (range 63-90). The average functional score improved from a mean of 32 points (range 10-65) to 72 points (range 40-90). The postoperative mechanical axis ranged between 3° of varus and 3° of valgus. There was an implant revision in one patient who had a traumatic rupture of medial collateral ligament, which occurred 27 months after the index procedure., Conclusions: Based on our results we think that the navigation-assisted technique provides an alternative approach to the traditional instrumentation for treating these difficult patients in an effective and less invasive manner.

Bone stock deficiency in primary as well as in revision total knee arthroplasty (TKA) represents a difficult problem to surgeon with regard to maintaining proper alignment of the implant components and in establishing a stable bone-implant interface. Different surgical procedures are available in these situations, for instances the use of bone cement, prosthetic augments, custom implant, and wire mesh with morsellized bone grafting and structural bone allograft. Structural allograft offers a numerous advantages as easy remodeling and felling cavitary or segmental defects, excellent biocompatibility, bone stock restoration and potential for ligamentous reattachment. In this article we report a short term result of three cases affected by severe segmental medial post/traumatic tibial plateau defect in arthritic knee, for which massive structural allograft reconstruction and primary total knee replacement were carried. The heights of the bone defect were between 27-33 mm and with moderate medio-lateral knee instability. Pre-operative AKS score in three cases was 30, 34 and 51 points consecutively and improved at the last follow-up to 83, 78 and 85 consecutively. No acute or chronic complication was observed. Last radiological exam referred no signs of prosthetic loosening, no secondary resorption of bone graft and well integrated graft to host bone. These results achieved in our similar three cases have confirmed that the structural bone allograft is a successful biological material to restore hemi-condylar segmental tibial bone defect when total knee replacement is indicated.

Background: two-stage revision is considered the best treatment approach for the eradication of chronic joint infection. We report the outcome of 41 consecutive patients with infected hip prostheses, treated between 2000 and 2005, with two-stage revision using an antibiotic-loaded cement spacer., Methods: Patients underwent a treatment protocol which included clinical and radiographic evaluation, laboratory investigations, hip aspiration, 99mTc-MDP and 99mTc-leukocyte-labeled scintigraphy and intraoperative assessment. All patients were diagnosed with a late chronic infection and classified as B-host according to the Cierny-Mader classification system. 9 patients out of 41 (22%) required a second interim treatment period, with exchange of the spacer. The proportion of methicillin-resistant Staphylococcus was similar between the one-spacer group and two-spacer group (28% vs 33%), whereas the proportion of patients with three or more risk factors was significantly higher in the two-spacer group than in the one-spacer group (28% vs 55%, respectively)., Results: Forty patients had final reimplantation, one patient had a resection arthroplasty. At an average follow-up of 5.3 years no recurrence of infection occurred. The average post-operative Harris hip score improved from 41 to 80., Conclusions: In the treatment of two-stage revision arthroplasty the adherence to the protocol proved to be effective for infection eradication and final reimplantation.

Benzidine (Bz) is a known human carcinogen. Several azo dyes have been synthesized with Bz. Bz can be metabolically released from azo dyes. In a group of Indian workers producing Bz and azo dyes the presence of hemoglobin (Hb) adducts was investigated. The following Hb adducts were identified and quantified by GC-MS: Bz, N-acetylbenzidine (AcBz), 4-aminobiphenyl (4ABP), aniline. 4ABP and aniline were quantitatively the major adducts. In the exposed workers (n = 33) all correlations between 4ABP, Bz and AcBz were r = 0.89 (P < 0.01) or greater. The group of workers exposed to Bz (Bz workers, n = 15) had 10-17-fold higher adduct levels than the workers exposed to dyes (dye workers, n = 18). 4ABP can be metabolically released from Bz and azo dyes. Aniline can be metabolically released from azo dyes. Therefore, the presence of 4ABP and aniline as Hb adducts is a consequence of exposure to the parent compounds or to the exposure of Bz and azo dyes and a consequent metabolical release of the arylamine moiety. The mean adduct ratios of 4ABP/(AcBz + Bz) varied up to 4-fold across all seven factories. Therefore, it is possible that 4ABP may have derived from general contamination in the work environment or endogenous metabolism, or a combination of the two. Since 4ABP is also a known human carcinogen, tumors observed in workers exposed to Bz or Bz dyes might be caused by both compounds. Further, these results suggest that understanding the role that genetic variants in NAT1 and NAT2 play in modifying the impact of Bz on bladder cancer risk may be complicated, as N-acetylation detoxifies 4ABP and activates Bz.

Nitrotoluenes, such as 2-nitrotoluene, 2,4-dinitrotoluene (24DNT), and 26DNT, are carcinogenic in animal experiments. Humans are exposed to such chemicals in the workplace and in the environment. It is therefore important to develop methods to biomonitor people exposed to nitrotoluenes to prevent the potential harmful effects. For the present study, workers exposed to high levels of these chemicals were investigated. The external dose (air levels), the internal dose (urine metabolites), the biologically effective dose [hemoglobin (Hb) adducts and urine mutagenicity], and biological effects (chromosomal aberrations and health effects) were determined. Individual susceptibility was assessed by determining genetic polymorphisms of enzymes assumed to function in nitrotoluene metabolism, namely glutathione S-transferases (GSTM1, GSTT1, GSTP1), N-acetyltransferases (NAT1, NAT2), and sulfotransferases (SULT1A1, SULT1A2). The levels of urinary metabolites did not correlate with the air levels. The urinary mutagenicity levels determined in a subset of workers correlated with the levels of a benzylalcohol metabolite of DNT. The Hb-adducts correlated with the urine metabolites but not with the air levels. The frequency of chromosomal aberrations (gaps included) was increased (P < 0.05) in the exposed workers in comparison with a group of factory controls and correlated with the level of 24DNT Hb-adducts in young subjects (<31 years). The GSTM1-null genotype was significantly more prevalent in the controls than in the exposed group, which probably reflected an elevated susceptibility of the GSTM1-null genotype to adverse health effects of DNT exposure, such as nausea (odds ratio, 8.8; 95% confidence interval, 2.4-32.2). A statistically significant effect was seen for SULT1A2 genotype on a 24DNT Hb-adduct; GSTP1 genotype on a 2,4,6-trinitrotoluene Hb-adduct; and SULT1A1, SULT1A2, NAT1, GSTT1, and GSTP1 genotypes on chromosomal aberrations in the exposed workers.

2,4,6-Trinitrotoluene (TNT) is an important occupational and environmental pollutant. In TNT exposed humans, the notable toxic manifestations have included aplastic anemia, toxic hepatitis, cataract, hepatomegaly and liver cancer. Therefore, we developed methods to biomonitor workers exposed to TNT. The workers were employed in a typical ammunition factory in China. The controls were recruited from the same factory. We determined hemoglobin (Hb) adducts and urine metabolites of TNT. Hb-adducts of TNT, 4-amino-2,6-dinitrotoluene (4ADNT) and 2-amino-4,6-dinitrotoluene (2ADNT), and the urine metabolites of TNT, 4ADNT and 2ADNT were found in all the workers and in a few controls. 4ADNT was the main product. Although the levels of 2ADNT correlated well with 4ADNT, 2ADNT was not found in all the samples. Therefore, 4ADNT was the best marker of exposure for Hb-adducts and urine metabolites. The levels of the urine metabolites and Hb-adducts were related to the health status of the workers. The Hb-adduct 4ADNT was statistically significantly associated with risk of hepatomegaly, splenomegaly and cataract. The odds ratio (OR) for cataract, splenomegaly and hepatomegaly were 6.4 [95% confidence interval (CI) = 1.4-29.6], 9.6 (1.1-85.3) and 7.6 (1.3-43.7), respectively. No correlation was found between urine metabolites and health effects. These results were tested for confounding factors like age, workyears, smoker status, smoke years, cigarettes per day and hepatitis B status using stepwise forward logistic regression analysis. In the case of splenomegaly, hepatitis B status is a confounder. In the case of cataract, age is a confounder. The Hb-adduct, 4ADNT, is a good biomarker of exposure and biomarker of biological effect.

Nitrotoluenes are important intermediates in the chemical industry. 2,6-Dinitrotoluene (26DNT), 2,4-dinitrotoluene (24DNT) and 2-nitrotoluene are carcinogenic in animals and possibly carcinogenic in humans. It is therefore important to develop methods to biomonitor workers exposed to such chemicals. Hemoglobin (Hb) adducts of nitroarenes are established markers of the biological effective dose. We developed a method to measure Hb adducts in biological samples. Hb adducts were measured in rats after a single exposure (0.5 mmol/kg) of 24DNT, 26DNT, 2,4-toluenediamine (24TDA) and 26TDA. Hydrolysis of Hb from rats dosed with 24DNT yields, 4-amino-2-nitrotoluene (4A2NT) (16.3 nmol/g Hb), 24TDA (4.3 nmol/g Hb) and 4-acetylamino-2-aminotoluene (4AA2AT) (0.51 nmol/g Hb). Hydrolysis of Hb from rats dosed with 26DNT yields three amines, 2-amino-6-nitrotoluene (2A6NT) (2.5 nmol/g Hb), 26TDA (1.2 nmol/g Hb) and 2-acetylamino-6-aminotoluene (2AA6AT) (0.17 nmol/g Hb). A similar Hb adduct pattern was found in Chinese workers exposed to nitrotoluenes. With respect to 24DNT, 4A2NT was the predominant adduct, and the amount was approximately 24-fold higher than 24TDA. With respect to 26DNT, 2A6NT was the predominant adduct, and the amount was approximately 20-fold higher than 26TDA. With respect to the mononitrotoluenes, the Hb adduct of 2NT was present in the highest concentrations. Each worker was examined for adverse health effects linked to exposure to DNT. The health effects were compared with the Hb adduct levels using logistic regression analysis. The odds of suffering from inertia were 3.2 times higher [95% confidence interval (CI) = 1.8-5.8] when the level of 4A2NT Hb adducts increased by one log-unit. Similar odds ratios were observed with somnolence (3.1, CI = 1.4-6.9), nausea (2.4, CI = 1.3-4.3) and dizziness (5.5, CI = 1.3-24.2). These results inferred that quantification of DNT-Hb adducts provided an effective biomarker of toxicity and could be used to estimate the risk associated with a particular exposure to DNT.

Background: In April 1991, an excess of bladder cancer cases among workers employed at a chemical manufacturing facility in Niagara Falls, NY, was reported. This excess was primarily confined to 708 workers who had ever been employed in the rubber chemicals manufacturing area of the plant, where the aromatic amines aniline and o-toluidine have historically been used., Purpose: An environmental and biological monitoring survey was conducted to evaluate current exposures to aniline and o-toluidine in the rubber chemicals department., Methods: Personal air sampling for aniline and o-toluidine was conducted with the use of a modified Occupational Safety and Health Administration (OSHA) 73 method. Urine samples were collected before and after work (i.e., pre-shift and post-shift, respectively) and stored at -70 degrees C. Base hydrolysis was used to convert acetanilide and N-acetyl-o-toluidine, metabolites of aniline and o-toluidine present in the urine, to the parent compounds. The parent compounds were extracted from the alkaline urine into butyl chloride and then back-extracted from the butyl chloride into aqueous hydrochloric acid. An aliquot of each acidic extract was subjected to ion-interaction reversed-phase liquid chromatography with coulometric electrochemical detection. Hemoglobin (Hb) was extracted from blood and stored at -70 degrees C. For the measurement of adducts of aniline, o-toluidine, and 4-aminobiphenyl (4-ABP), precipitated Hb was dissolved in 0.1 M sodium hydroxide in the presence of recovery standards, and the hydrolysate was extracted with hexane, derivatized with pentafluoropropionic anhydride, and analyzed by gas chromatography-mass spectrometry with negative chemical ionization., Results: A total of 73 workers, including 46 of 64 exposed workers who were employed in the rubber chemicals department and had the potential for exposure to aniline and o-toluidine and 27 of 52 unexposed workers employed in other departments where aniline and o-toluidine were not used or produced, had data available for both aniline and o-toluidine and Hb adducts; 28 of the workers in the former group also had personal air-sampling data. Personal air sample measurements showed that airborne concentrations of aniline and o-toluidine were well within the limits allowed in the workplace by OSHA. Urinary aniline and o-toluidine levels, however, were substantially higher among exposed workers than among unexposed control subjects. The most striking differential was for post-shift urinary o-toluidine levels, which averaged (+/- standard deviation) 2.8 micrograms/L (+/- 1.4 micrograms/L) in unexposed subjects and 98.7 micrograms/L (+/- 119.4 micrograms/L) in exposed subjects (P = .0001). Average aniline-Hb and o-toluidine-Hb adduct levels were also significantly higher (P = .0001) among exposed workers than among unexposed control subjects. Average levels of adducts to 4-ABP, a potential contaminant of process chemicals, were not significantly different (P = .48), although three exposed workers had 4-ABP levels above the range in unexposed workers., Conclusions: The adduct data suggest that, among current workers, o-toluidine exposure substantially exceeds aniline exposure and that 4-ABP exposure, if it occurs at all, is not widespread. These data support the conclusion that occupational exposure to o-toluidine is the most likely causal agent of the bladder cancer excess observed among workers in the rubber chemicals department of the plant under study, although exposures to aniline and 4-ABP cannot be ruled out.

Aflatoxin is implicated as a risk factor for hepatocellular carcinoma in areas of the world with a high incidence of this tumor. The present study was designed to validate the use of aflatoxin-albumin adducts in peripheral blood as a measure of individual exposure to this carcinogen. Dietary intake of aflatoxin was measured at the individual level in 20 residents of Keneba, West Kiang, The Gambia, over a 7-day period and correlated with the level of aflatoxin bound to peripheral blood albumin at the beginning and end of the study. Complementary enzyme-linked immunosorbent assay and high-performance liquid chromatography-fluorescence techniques were used to assay the aflatoxin adducts. All subjects were exposed to aflatoxin originating from several food types, with an average daily intake of 1.4 micrograms/day. A significant correlation (r = 0.55; P = < 0.05) was observed between the dietary intake and the level of albumin-bound aflatoxin at the end of the study. In addition, a good agreement was obtained with the two analytical techniques. A comparison of matched chronic hepatitis B surface antigen carriers with noncarriers did not reveal any difference in adduct formation for a given dietary intake of aflatoxin. These studies demonstrate the validity of aflatoxin-albumin adducts as a marker of human exposure to this carcinogen.

Aromatic amines are important intermediates in industrial manufacturing. N-oxidation to the N-hydroxyarylamines is a key step determining the genotoxic properties of aromatic amines. N-hydroxyarylamines can form adducts with DNA, with tissue proteins and with the blood proteins albumin and hemoglobin in a dose-dependent manner. The determination of hemoglobin adducts is a useful tool for biomonitoring exposed populations. We have established the hemoglobin binding index (HBI) [(mmol compound/mol Hb)/(mmol compound/kg body wt)] of several aromatic amines in female Wistar rats. Including the values obtained by other researchers in the same rat strain, the logarithm of hemoglobin binding (log HBI) was plotted against the following parameters: the sum of the Hammett constants (sigma sigma = sigma p + sigma m), pKa, log P (octanol/water), the half wave oxidation potential (E1/2) and the electronic descriptors of the amines and their corresponding nitrenium ions obtained by semiempirical calculations (MNDO, AM1 and PM3), such as atomic charge densities, energies of the HOMO and LUMO and their coefficients, the C-N bond order, the dipole moments and the 'reaction enthalpy' [MNDOHF, AM1HF or PM3HF = Hf(nitrenium) - Hf(amine)]. The correlation coefficients were determined from the plots of all parameters against log HBI for all amines by means of linear regression analysis. The amines were classified into three groups: group 1, all para-substituted amines, group 2, all amines with halogens and group 3, all amines with alkyl groups. For the amines of group 1, log HBI correlates with sigma sigma, MNDOHF, E1/2, the pKa and the log P with r = 0.84, 0.71, 0.73, - 0.69 and 0.50, respectively. For the amines of group 2, log HBI correlates with pKa, sigma sigma, MNDOHF, E1/2 and log P with r = 0.81, -0.76, -0.55, -0.46 and -0.20, respectively. For the amines of group 3, log HBI correlates with the E1/2, PM3HF, sigma sigma, pKa and log P with r = 0.92, 0.89, 0.76, 0.19 and 0.12, respectively. The apparent Michaelis-Menten constants Km and Vmax of the N-acetyltransferase of liver cytosol were determined for several amines. Km and Vmax do not correlate with any of the electronic descriptors. Female Wistar rats were dosed with nitroarenes. Hemoglobin binding of nitroarenes correlates with the energy levels of the LUMO. This investigation determines for a large variety of aromatic amines the bioavailability of the N-hydroxyarylamine--the genotoxic metabolite--and the utility of electronic descriptors for prediction of the N-oxidation.

Aflatoxin (AF) albumin adducts are found in peripheral blood after exposure to aflatoxin B1 (AFB1) and the measurement of these adducts is potentially a useful tool in the epidemiological study of the role of AFB1 in the etiology of liver cancer. Three complementary approaches to the quantitation of AF-albumin adducts are described: (a) enzyme-linked immunosorbent assay (ELISA) performed directly on intact albumin (direct ELISA); (b) ELISA performed on an albumin hydrolysate (hydrolysis ELISA); (c) high-performance liquid chromatographic fluorescence detection of AF-lysine adduct after albumin hydrolysis and immunoaffinity purification. These techniques have been validated by direct comparison with rat albumin samples modified to a known extent. Detection limits of approximately 100, 5.0, and 5.0 pg AF/mg human albumin were determined for the three methods, respectively. Samples obtained from individuals from Thailand, The Gambia, Kenya, and France have been used to validate the measurement of AF-albumin adducts by these three methods. Levels of 7 to 338 pg AF/mg albumin were observed in the former two countries while no adducts were detected in samples from France. The relative properties of the three assays, with special regard to their application in epidemiological studies, are considered. A combination of the hydrolysis ELISA for large scale screening followed by confirmatory analyses in positive samples by high-performance liquid chromatographic fluorescence is suggested as an optimum methodology.

Aflatoxin B1 (AFB1) is a potent carcinogen. It reacts with liver DNA and serum albumin in a dose-dependent manner. Serum albumin adducts of aflatoxins have been used for exposure assessment. The immunological methods used so far do not differentiate between the different adducts of AFB1 and of AFG1. In order to establish an analytical method to measure one specific AFB1 adduct, we investigated the structure and the chemistry of the major serum albumin adduct of aflatoxin B1 (lysine-AFB1). 13C-NMR, 1H-NMR, UV, IR, fluorescence and MS spectra of lysine-AFB1 (8-[N-(2-amino-hexanoyl-6-yl)-5-oxo-3-pyrrolin-3-yl]-7-hydroxy-5- methoxycyclopentenone[2.3-c]coumarin) were recorded and discussed. The quantification of lysine-AFB1 was demonstrated in biological samples. Serum albumin was digested with pronase and analysed by HPLC with a fluorimeter as detector. The detection limit found for lysine-AFB1 was 20 fmol.