Radiological assessment of the PRF/BMSC efficacy in the treatment of aseptic nonunions: A retrospective study on 90 subjects.

BACKGROUND: Nonunion is a major orthopaedic concern because of treatment difficulty, high costs and devastating effects on the patients' life quality. Therefore, there is interest in the use of bone substitutes and cell-based strategies to augment fracture repair. We aimed to verify if Platelet Rich Fibrin (PRF) added with bone marrow stromal cells (BMSC) was able to improve the reparative process in the aseptic nonunion, and to establish whether it was worthwhile with atrophic nonunion. The primary outcome was radiological union. As secondary endpoint, the healing time was assessed, and the radiological consolidation grade at each follow-up. METHODS: We identified 113 subjects with tibia or femur nonunion and retrospectively created two groups. Group A was constituted by 56 subjects who underwent the standard procedure, i.e. Judet decortication with/out internal fixation devices, and opposite cortical homoplastic stick. In 57 patients, the standard procedure was modified by adding PRF and BMSC carried by homologous lyophilised bone chips (group B). The same surgeon performed all the operations. To our knowledge, no data are reported in the literature about such application. Since a "gold standard" for healing quantification does not exist, a new scoring radiological system was applied, at 1.5, 3, 6, 12 and 24 months after treatment. RESULTS: At the final 24-month follow-up, the radiological union percentage was 94,12 in group B and 95,12% in group A. A decreased healing time was demonstrated in the presence of PRF/BMSC in comparison with the standard procedure. When we compared the radiological scores at each follow-up, we found that the PRF/BMSC combination significantly improved the consolidation grade at 1.5-, 3- and 6-month follow-up in femurs and at 1.5-month follow-up in tibiae. Furthermore, an improved consolidation grade was demonstrated in the atrophic subjects treated with adjuvants compared to atrophic patients treated with the standard procedure at 1.5-month follow-up. CONCLUSIONS: This study supports the concept that the use of PRF/BMSC, during the standard procedure, is effective in shortening nonunion healing time. It could allow an early mobilization of patients, minimizing suffering, and could be an effective tool to reduce the health-care costs resulting from this issue. LEVEL OF EVIDENCE: Therapeutic level III.

Total knee arthroplasty for post-traumatic proximal tibial bone defect: three cases report.

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.

Gap balancing versus measured resection technique using a mobile-bearing prosthesis in computer-assisted surgery.

Navigation has been developed to help surgeons install implants more accurately and reproducibly; at the same time, this tool is able to record quantitative information such as joint range of motion, laxity and kinematics intra-operatively. As for standard surgery, two strategies are possible to achieve either femoral component rotation or overall prosthetic alignment: a measured gap resection approach, in which bone landmarks are used to guide resections equal to the distal and posterior thickness of the femoral component, or a gap-balancing technique, in which equal collateral ligament tension in flexion and extension is tried to find before as a guide to final bone cuts. The purpose of this paper is to compare the two different methods in a 67 patients group submitted to the same procedure using mobile-bearing (MB) prosthesis in order to analyse the effect of both techniques on joint line maintenance, axial limb restoration and components position. The gap group (GG) consists of 31 patients in whom the arthroplasty was performed using a navigated gap-balancing technique. The measured group (MG) consists of 36 patients in whom a computer-assisted measured resection technique was used. The results of imaging and the number of outliers were not statistically different (P = 0.56) for the mechanical axis and prosthetic positioning between the two groups. The gap technique showed a statistically significant alteration of the post-operative value when compared with the measured resection technique, (P = 0.036). The mean elevation of the joint line was 4.09 mm for the GG and 3.50 mm in the MG.

Comparison between two computer-assisted total knee arthroplasty: gap-balancing versus measured resection technique.

Two surgical strategies are possible in total knee arthroplasty (TKA): a measured resection technique, in which bone landmarks are used to guide resections equal to the distal and posterior thickness of the femoral component, or a gap-balancing approach, in which equal collateral ligament tension in flexion and extension is sought before and as a guide to final bone cuts. In this study performed with computer assisted system, we compared the 2 different methods in 126 patients followed prospectively in order to analyze the effect of both the techniques on joint-line (JL) maintenance, axial limb restoration and components position. The gap technique showed a statistical increase in the post-operative value when compared with the measured resection technique, (P = 0.008). When comparing the two groups regarding to the pre-operative deformity, we have found a statistical difference (P = 0.001) in case of moderate pre-operative deformity (less than 10 degrees), and the measured resection technique showed a slight superiority in preserving a joint line more faithful to the pre-operative. We found an ideal alignment for the mechanical axis (180 degrees ± 3 degrees) (95% of cases). In six cases (5%), the mean post-operative value exceeded (varus or valgus) the ideal value by more than 3 degrees. In the frontal plane, a good alignment was observed for both femoral and tibial components without a significant difference between the two techniques. In the sagittal plane was found more alignment variability due to the different implants used and their ideal starting slope, from 7 degrees to 3 degrees. Finally, the surgeon can use the approach with which he has more confidence; however, as the measured resection technique causes less reduction in the post-operative joint-line position, in case of shortening of patellar tendon or patella infera, this technique is preferable.

Chronic recurrent multifocal and aspecific osteomyelitis: a case report.

Chronic recurrent multifocal and unspecific osteomyelitis (CRMUO) is a severe form of chronic nonbacterial osteomyelitis. Lesions can be found anywhere in the skeleton, in young patients, such as children or adolescents, chronic nonbacterial osteomyelitis often affects the metaphyses of the long bones. Furthermore, other organs like skin, eyes and gastrointestinal tract can also be affected. Because of symptoms and course of disease vary in significant way, a clinical diagnosis is often difficult. The radiographic appearance suggests subacute or chronic osteomyelitis. CRMUO may mimic acute hematogenous osteomyelitis, but bacterial culture are usually negative and nonspecific histopathological and laboratory findings are present. This kind of osteomyelitis is often diagnosed by exclusion of the two main differential diagnoses-bacterial infections and tumor-by assessing for a characteristic course and the findings by conventional radiography, if necessary supplemented by scintigraphy and magnetic resonance imaging (MRI). The MRI appearance of CRMUO lesions in tubular bones and the spine is often rather characteristic and can support the diagnosis. It is important to diagnose CRMUO to avoid unnecessary diagnostic procedures or therapy, and initiate an appropriate one. We present a case report of a 44-year-old woman diagnosed with CRMUO involving both femurs.

Early aseptic loosening of a porous tantalum knee prosthesis.

Porous tantalum represents a relatively new solution for primary and revision total knee arthroplasty, offering several unmatched properties. Tantalum is a transition metal, with excellent biocompatibility and bioactivity due to its intrinsic physical and structural characteristics. A widespread clinical use of porous tantalum tibial components for primary total knee arthroplasty has been partly hindered by the difficulty in removing this type of implant after bone in growth, often leading to a significant bone defect. On the contrary, in the case here reported, removal of the trabecular metal tibial component was unexpectedly easy at a 7-month follow-up due to the absence of bone ingrowth but with a complete preservation of the tibial plate bone stock. Causes for the lack of bone ingrowth are discussed.

Preliminary experience with electromagnetic navigation system in TKA.

Accuracy of implant positioning and precise reconstruction of leg alignment offers the best way to achieve good long-term results in total knee arthroplasty. Computer instrumentation was developed to improve the final position of the component and restore the mechanical axis. Current navigation systems use either optical or electromagnetic tracking. The advantage of the Electromagnetic (EM) navigation system is that no line-of-sight issues are present. However, special iron-free instruments are required. This report analyzes the postoperative radiological results of 32 knees treated using an EM system. All the measurements were recorded using software able to subtend angles automatically by five physicians, three radiologist and two orthopedic residents not involved with the surgery. Each radiograph was measured three times, in random order, and at delayed intervals. We found an ideal alignment for the mechanical axis (180+/-3 degrees ) in 30 out of 32 cases, whereas all the patients achieved a value of 90 degrees +/-3 degrees for both femoral and tibial frontal component angles. An apparently over-corrected implant position for the sagittal femoral component was reported, with a mean value of 11.2 degrees +/-3.6. The mean position of the tibial component was 90.6 degrees +/-2.8; just four measurements were outside of the +/-3 degrees of the desired value. EM is safe and there were no complications related to this system. An almost perfect correlation was found between the mechanical axis value of the EM navigation system (179.8 degrees +/-1.8) and the median value of the all reviewers (180.3 degrees +/-1.9) with a difference of 0.5 degrees .

Trabecular metal patella in total knee arthroplasty with patella bone deficiency.

This study evaluates results following patellar resurfacing using trabecular metal (TM) patella in marked deficiency or weakness of patellar bone that precludes patellar resurfacing with a standard cemented patellar button. Ten consecutive patients undergoing primary (3 cases) or revision (7 cases) total knee arthroplasty with patella augmentation were evaluated at a mean follow-up of 45 months (range 18-65). Nine patients had marked patellar bone deficiency and one had had previous patellectomy. No intra-operative complications occurred. There was no displacement of the patellar component and no patellar fractures when at least 50% of bone contact was possible. We observed loosening of the patella augmentation 17 months after the index procedure only in the case of previous patellectomy. When bone was present the fixation appeared excellent by radiographic evaluation already at 3 to 6 months after surgery; afterward bone contact was uniform in the peripheral regions in both lateral and Merchant radiographic views without signs of loosening. Finally, the mean Knee Society scores improved in all patients.

Urinary metabolites and health effects in workers exposed chronically to chloronitrobenzene.

For workers exposed to 4-chloronitrobenzene (4CNB), the major metabolites were determined. Urine were analysed before and after acid hydrolysis to qualify the free and conjugated metabolites of 4CNB. Three conjugated metabolites were identified in exposed workers: the mercapturic acid N-acetyl-S-(4-nitrophenyl)-L-cysteine (NANPC) was the only metabolite detected in non-hydrolysed urine, and accounted for approximately 51% of the total metabolites detected. The two remaining metabolites 4-chloroaniline (4CA) and 2-chloro-5-nitrophenol (CNP) were identified as cleavage products in hydrolysed urine, and accounted for approximately 18 and 30% of the total metabolites detected, respectively. No metabolites were found in factory controls within the limits of quantitation (LOQ) of the assay. There is a moderate correlation between NANPC and both 4CA and CNP. The correlation between 4CA and CNP is minor. The correlation between the total metabolites and both 4CA and CNP are good. The best correlation was found between the total metabolites and NANPC. There is a moderate inverse correlation between age and the creatinine levels. The raw metabolite levels CNP and NANPC decrease with age. The urine metabolites increase and correlate significantly with the creatinine levels. 4CA, NANPC and the total metabolite levels correlate with the haemoglobin adduct levels. NANPC is the most appropriate biomarker in the urine for a recent absorbed dose of 4CNB, since NANPC reflects the levels of 4CA and CNP and is the most prevalent metabolite detected in all the exposed workers.

Comparison of biomarkers in workers exposed to 2,4,6-trinitrotoluene.

2,4,6-Trinitrotoluene (TNT) is an important occupational and environmental pollutant. In TNT-exposed humans, notable toxic manifestations have included aplastic anaemia, toxic hepatitis, cataracts, hepatomegaly, and liver cancer. Therefore, methods were developed to biomonitor workers exposed to TNT. The workers were employed in a typical ammunition factory in China. The external dose (air levels and skin exposure), the internal dose (urinary metabolites), the biologically effective dose (haemoglobin adducts, urinary mutagenicity), biological effects (chromosomal aberrations and health effects), and individual susceptibility (genotypes of xenobiotic-metabolizing enzymes) were determined. Haemoglobin-adducts of TNT, 4-amino-2,6-dinitrotoluene (4ADNT) and 2-amino-4,6-dinitrotoluene (2ADNT), and the urinary metabolites of TNT, 4ADNT and 2ADNT, were found in all workers and in some controls. The levels of the haemoglobin-adducts or the urinary metabolites correlated weakly with the skin or air levels of TNT. The urinary mutagenicity determined in a subset of workers correlated strongly with the levels of 4ADNT and 2ADNT in urine. The haemoglobin-adducts correlated moderately with the urinary metabolites and with the urinary mutagenicity. The genotypes of glutathione S-transferases (GSTM1, GSTT1, GSTP1) and N-acetyltransferases (NAT1, NAT2) were determined. In general, the genotypes did not significantly influence the haemoglobin-adduct levels and the urine metabolite levels. However, TNT-exposed workers who carried the NAT1 rapid acetylator genotype showed an increase in urinary mutagenicity and chromosomal aberrations as compared with slow acetylators. The haemoglobin adduct 4ADNT was significantly associated with a risk of hepatomegaly, splenomegaly and cataract; urine metabolites and genotypes were not associated with health effects. These results indicate that a set of well-selected biomarkers may be more informative regarding exposure and effect than routinely performed chemical measurements of pollutants in the air or on the skin.

Urinary metabolites of workers exposed to nitrotoluenes.

Nitrotoluenes are important intermediates in the chemical industry. 2,6-Dinitrotoluene (26DNT), 2,4-dinitrotoluene (24DNT) and 2-nitrotoluene (2NT) are carcinogenic in animals and possibly carcinogenic in humans. Thus, it is important to develop methods to biomonitor workers exposed to such chemicals. The authors have monitored the air and urine metabolite levels for a group of workers in China exposed to 24DNT, 26DNT, 2NT and 4-nitrotoluene (4NT). The metabolites 2,4-dinitrobenzylalcohol (24DNBAlc), 2-amino-4-nitrobenzoic acid (2A4NBA), 4-amino-2-nitrobenzoic acid (4A2NBA) and 2,4-dinitrobenzoic acid (24DNBA) resulting from exposure to 24DNT were found in 89, 88, 91 and 78% of the exposed workers, respectively. The metabolites 2,6-dinitrobenzylalcohol (26DNBAlc) and 2,6-dinitrobenzoic acid resulting from 26DNT exposure were found in 99 and 86% of the exposed workers, respectively. Quantitatively, 2A4NBA, 4A2NBA and 26DNBAlc were the major metabolites. The nitrobenzoic acids were the major metabolites resulting from exposure to 2NT and 4NT and were present in 96 and 73% of the exposed workers, respectively. Air concentrations of DNT and 2NT did not correlate with the levels of metabolites in the urine. In conclusion, the dinitrobenzyl alcohols and aminonitrobenzoic acids determined in the urine provided a good marker for recently absorbed dose and were intrinsically related to the bioactivation and detoxification pathways of DNT. Air measurements were not a good measure to predict internal exposure.

Synthesis of adducts with amino acids as potential dosimeters for the biomonitoring of humans exposed to toluenediisocyanate.

Toluenediisocyanates (TDI) are important intermediates in the chemical industry. Among the main damages after low levels of TDI exposure are lung sensitization and asthma. Protein adducts of TDI might be involved in the etiology of sensitization reactions. Blood protein adducts are used as dosimeters for modifications of macromolecules in the target organs where the disease develops. The functional groups of cysteine, tyrosine, serine, lysine, tryptophan, histidine, and N-terminal amino acids are potential reaction sites for isocyanates. Especially the N-terminal amino acids, valine, and aspartic acid of hemoglobin and albumin, respectively, are reactive toward electrophilic xenobiotics. To develop methods for the quantitation of protein adducts of 2,4- and 2,6-TDI, we reacted 3-nitro-4-methylphenyl isocyanate (1a) with single amino acids and reduced the nitro group using catalytic hydrogenation or ammonium formate with palladium on carbon yielding N-[(3-amino-4-methylphenyl)carbamoyl]valine (2a), N-[(3-amino-4-methylphenyl)carbamoyl]aspartic acid (8a), N(alpha)-acetyl-N(epsilon)-[(3-amino-4-methylphenyl)carbamoyl]lysine (12a), and N(alpha)-acetyl-O-[(3-amino-4-methylphenyl)carbamoyl]serine (15a). The same reactions were performed with 5-nitro-2-methylphenyl isocyanate (1b) and 3-nitro-2-methylphenyl isocyanate (1c). The valine adducts were boiled in acid to obtain the corresponding hydantoins: 3-(3-amino-4-methylphenyl)-5-isopropylimidazoline-2,4-dione (5a), 3-(5-amino-2-methylphenyl)-5-isopropylimidazoline-2,4-dione (5b), and 3-(3-amino-2-methylphenyl)-5-isopropylimidazoline-2,4-dione (5c). A method for the detection of N-terminal adducts with valine in biological samples was developed. The tripeptide adduct N-[(3-amino-4-methylphenyl)carbamoyl]valyl-glycyl-glycine (19a) was hydrolyzed with acid in the presence of globin and the internal standard N-[(3-amino-4-methylphenyl-d(6))carbamoyl]valyl-glycyl-glycine (19d). The released hydantoins were determined by LC/MS/MS and after derivatization with pentafluoropropionic anhydride by GC/MS. The determination limit was 0.16 pmol/sample. The same N-terminal adduct with valine was found in globin of a TDI-worker and in two women with polyurethane covered breast implants.

Carcinogenic risk of toluene diisocyanate and 4,4'-methylenediphenyl diisocyanate: epidemiological and experimental evidence.

Diisocyanates are highly reactive compounds widely used, for example, in the production of polyurethane foams, elastomers, paints, and adhesives. The high chemical reactivity of these compounds is also reflected in their toxicity: diisocyanates are one of the most important causes of occupational asthma but also other adverse effects, such as irritation and toxic reactions, have been described in exposed subjects. One of the open questions is whether occupational isocyanate exposure is a carcinogenic hazard. The few epidemiological studies available have been based on young cohorts and short follow-up and are not conclusive. Toluene diisocyanate (TDI) has been classified as carcinogenic in animals on the basis of gavage administration studies, but no conclusions are available on inhalation exposure. For 4,4'-methylene diphenyldiisocyanate (MDI) there is suggestive evidence for carcinogenicity in rats. The possible carcinogenic mechanism of TDI and MDI is not clear. Both chemicals have been positive in a number of short-term tests inducing gene mutations and chromosomal damage. The reactive form could be either the diisocyanate itself or may derive from the metabolic activation of the aromatic diamine derivatives formed by hydrolysis. TDI and MDI react with DNA in vivo and in vitro. However, the structure of the adducts has not been identified. Especially from the in vivo experiment it is not known if the adducts are a product from the reaction with the isocyanate or the corresponding amine. In conclusion, both TDI and MDI are highly reactive chemicals that bind to DNA and are probably genotoxic. The alleged animal carcinogenicity of TDI and MDI would suggest that occupational exposure to these compounds is a carcinogenic risk. The few epidemiological studies available have not, however, been able to clarify if TDI and MDI are occupational carcinogens.

Haemoglobin adducts of aromatic amines: diamines and polyaromatic amines.

Aromatic amines and nitroarenes are important antioxidants and intermediates in the synthesis of dyes, pesticides and plastics. In the present paper we introduce methods for the synthesis of deuterated standards: 3-[2H8]aminofluoranthene, 3,3'-dimethyl-[2H4]benzidine, [2H4]benzidine, N'-acetyl-[2H4]benzidine, 2,4-[2H6]toluenediamine, 2,6-[2H6]toluenediamine. These standards have been used for the quantification of haemoglobin adducts of diamines and polyaromatic amines. Haemoglobin was hydrolysed in 0.1 M sodium hydroxide and the hydrolysate extracted with dichloromethane. The extracts were derivatised with heptafluorobutyric anhydride and analysed by GC-MS with negative chemical ionisation. In one run up to 15 aromatic amines can be determined: 6-aminochrysene, 3-aminofluoranthene, 2-aminofluorene, 1-aminopyrene, benzidine, 3,3'-dichlorobenzidine, 3,3'-dimethoxybenzidine, 3,3'-dimethylbenzidine, 3,3'-methylenedianiline, 4,4'-methylenedianiline, N'-acetyl-benzidine, N'-acetyl-4,4'-methylenedianiline, 4,4'-methylene bis(2-chloroaniline), 2,4-toluenediamine and 2,6-toluenediamine.

Isocyanate-specific hemoglobin adduct in rats exposed to 4, 4'-methylenediphenyl diisocyanate.

4,4'-Methylenediphenyl diisocyanate (MDI) is the most important of the isocyanates used as intermediates in the chemical industry. Among the main types of damage after exposure to low levels of MDI are lung sensitization and asthma. Protein adducts of MDI might be involved in the etiology of sensitization reactions. It is therefore necessary to have sensitive and specific methods for monitoring the isocyanate exposure of workers. To date, urine metabolites or protein adducts have been used as biomarkers in workers exposed to MDI. However, with these methods it is not possible to determine if the biomarkers result from exposure to MDI or to the parent aromatic amine 4,4'-methylenedianiline (MDA). This work presents a procedure for quantitating isocyanate-specific hemoglobin adducts. Blood proteins are used as markers of exposure and possibly as markers of dose size for the modifications of macromolecules in the target organs where the disease develops. For the quantitation of hemoglobin adducts, N(1)-[4-(4-isocyanatobenzyl)phenyl]acetamide (AcMDI) was reacted with the tripeptide valyl-glycyl-glycine and with valine yielding N-[4-(4-acetylaminobenzyl)phenyl]carbamoyl]valyl-glycyl-glycine and N-[4-[4-(acetylaminobenzyl)phenyl]carbamoyl]valine, respectively. N-[4-[4-(Acetylamino-3,5-dideuteriobenzyl)-2, 6-dideuteriophenyl]carbamoyl]valine was synthesized from valine, as was N(1)-[4-(4-isocyanato-3,5-dideuteriobenzyl)-2, 6-dideuteriophenyl]acetamide, for use as an internal standard. These adducts were cleaved in 2 M HCl to yield the corresponding hydantoins, 3-[4-(4-aminobenzyl)phenyl]-5-isopropyl-1, 3-imidazoline-2,4-dione (MDA-Val-Hyd) and 3-[4-(4-amino-3, 5-dideuteriobenzyl)-2,6-dideuteriophenyl]-5-isopropyl-1, 3-imidazoline-2,4-dione, respectively. In globin of rats exposed to MDI, MDA-Val-Hyd could be found in a dose-dependent manner. The adduct was identified by HPLC/MS/MS and quantified by GC/MS after derivatization with heptafluorobutyric anhydride. The amount of MDA-Val-Hyd found after acid hydrolysis of globin at 100 degrees C is about 12 times larger than the sum of N-acetyl-4, 4'-methylenedianiline (AcMDA) and MDA obtained from mild base hydrolysis of hemoglobin. The MDA-Val-Hyd is an isocyanate-specific adduct. MDA and AcMDA released after mild base hydrolyses result most likely from a sulfinamide adduct which is a typical adduct of arylamines. According to these results, higher amounts of isocyanate adducts than arylamine adducts should be expected in workers exposed to isocyanates.

Hemoglobin binding of bicyclic aromatic amines.

Aromatic diamino compounds, e.g., 4,4'-methylenebis(2-chloroaniline) (MOCA) and 4,4'-methylenedianiline (MDA), are used as curing agents in the production of elastomers. Since MOCA and MDA are mutagenic and carcinogenic, substitutes are of great commercial interest. For benzidine it has been shown that ortho substitution with methyl groups yields the nonmutagenic 3,3',5,5'-tetramethylbenzidine. Therefore, MDA analogues with large substituents in the ortho position have been synthesized. The substituents are supposed to inhibit the formation of the N-hydroxyarylamines which are the putative genotoxic intermediates. We investigated the biological availability of the N-hydroxylamines of ortho-substituted diamines and of known carcinogenic diamines in female Wistar rats, by determining hemoglobin (Hb) adducts. Hb from rats dosed with 0.5 mmol/kg diamine and from controls was isolated and hydrolyzed in base. The released diamine and monoacetyldiamine were quantified by HPLC with electrochemical detection and/or GC/MS. MDA, 4,4'-oxydianiline (ODA), 4,4'-ethylenedianiline, and 4,4'-thiodianiline (TDA) bound to hemoglobin as diamine and as monoacetyl-diamine. 4,4'-Methylenebis(2,6-dimethylaniline), 4,4'-methylenebis(2,6-diethylaniline), MOCA, and 4,4'-sulfonyldianiline (dapsone) bound only as diamine to Hb. 4,4'-Methylenebis(2,6-dichloroaniline) did not bind to Hb. Thus, the presence of two substituents in the ortho position and the presence of electron-withdrawing groups in the para position to the amino group drastically reduced the formation of Hb adducts. The amount of hemoglobin adducts was compared to their carcinogenic potency. The extent of hemoglobin binding of the bicyclic diamines (dapsone, 3,3'-dichlorobenzidine, MDA, MOCA, TDA, ODA, and benzidine) increases with their carcinogenic potency.

Synthesis and quantification of DNA adducts of 4,4'-methylenedianiline.

4,4'-Methylenedianiline (MDA) is used as a hardener in the manufacture of plastics and polyurethanes. MDA has been classified as a carcinogen in animals and is a suspected human carcinogen. Assuming that MDA would yield similar DNA adducts to other arylamines, we synthesized the following C-8 guanine adducts: N'-acetyl-N-(deoxyguanosin-8-yl)-MDA, N-(deoxyguanosin-8-yl)-MDA, N-(deoxyguanosin-8-yl)-4MA, and their corresponding 3'-monophosphate derivatives. We developed methods to identify these adducts of MDA in liver DNA using 32P-postlabeling, HPLC, and GC-MS techniques. Liver DNA was obtained from rats treated with radiolabeled MDA (1.11 and 116.5 mumol/kg body weight). The total radioactivity bound to the DNA corresponded to 0.06 and 2.7 adducts per 10(7) nucleotides [covalent binding index (CBI = (mumol of adduct per mol of nucleotide)/(mmol of compound per kg body weight)) of 1.05 and 2.3]. This DNA-binding potency is in the range of weakly genotoxic compounds. The liver DNA was analyzed for the presence of the synthesized adducts by the following methods: (I) HPLC analysis of nucleotides and purines after enzymatic and acid hydrolysis, and (II) 32P-postlabeling after enzymatic hydrolysis. The major adducts found in vivo did not correspond to the synthesized standards. Further work was carried out to determine the structure of the unidentified adducts. It was possible to release MDA and MDA-d4 from DNA of rats dosed with MDA and/or MDA-d4 and from the synthesized adducts using strong base hydrolysis. Liver of two female Wistar rats given 500 mumol/kg MDA-2HCl was hydrolyzed in 0.1 M NaOH overnight at 110 degrees C. GC-MS analysis of the heptafluorobutyric anhydride derivatized dichloromethane extracts detected 428 +/- 40 fmol of MDA/mg of DNA. In the control animals no MDA was found. The experiment was repeated with livers from animals dosed 500 mumol/kg MDA-d4.2DCl. In these rats 488 +/- 19 fmol MDA-d4 was found to be bound at liver DNA. Taking into account a 68% yield of the method, the CBI found in these cases was 0.82 and 1.0, respectively.

Monitoring of aromatic amine exposures in workers at a chemical plant with a known bladder cancer excess.

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.

Determination of hemoglobin adducts in workers exposed to 2,4, 6-trinitrotoluene.

2,4,6-Trinitrotoluene (TNT) is an important occupational and environmental pollutant. TNT can be taken up through the skin and by inhalation. It is therefore essential to have fast and reliable methods to monitor human exposure. In rat experiments, it has been shown that TNT binds covalently to blood proteins and to tissue proteins. Hemoglobin (Hb) adducts of TNT are markers for the internal dose and possibly for the toxic effects of TNT, e.g. cataracts. In the present paper we introduce a new efficient method to quantify Hb adducts of TNT. Precipitated Hb was hydrolyzed with base in the presence of the surrogate internal standard 3,5-dinitroaniline (35DNA). The released 2-amino-4,6-dinitrotoluene (2ADNT) and 4-amino-2,6-dinitrotoluene (4ADNT) were quantified against 35DNA by gas chromatography-mass spectrometry with negative-ion chemical ionization. Hb of 50 workers and controls from a Chinese munition factory were investigated. The Hb adduct levels ranged from 3.7 to 522 ng for 4ADNT and from 0 to 14.7 ng for 2ADNT per gram of Hb. However, in control samples from Germany no Hb adducts of 4ADNT or 2ADNT could be found.