Off-label use of orthopedical trauma implants in a low-income country.

PURPOSE: Lack of resources, severe injuries, and logistical flaws force surgeons in low-income countries (LIC) to improvise during surgery and use implants "off-label." These off-label treatments are specific for the work of trauma surgeons in non-governmental (NGO) hospitals in LIC. The aim of this study is to show the need of off-label surgery in an environment of low resources by means of typical examples. METHODS: Off-label treated fractures, the implant used instead, and the reason for off-label treatment were investigated in 367 injuries over a three month period in an NGO hospital in Sierra Leone. RESULTS: Twenty-seven fractures were treated off-label with mostly K-wires (88.89%) and external fixators (51.85%). Three reasons for off-label use could be defined: no suitable implants (N = 14), the condition of soft tissues that did not allow internal osteosyntheses (N = 10), and implants not ready for surgery due to logistic flaws (N = 3). The implants needed were mostly locking plates. CONCLUSION: Surgeons in similar settings must use K-wires and external fixators to treat complex fractures. Using implants off-label can help surgeons to treat fractures otherwise left untreated.

No implant, no solution, lost cases to surgery: orthopedic trauma triage for surgery in an NGO hospital in Sierra Leone.

INTRODUCTION: In low-income countries (LIC), international surgeons face the fact that there are patients they cannot treat. The goal of this study was to identify and analyze patients lost to treatment. MATERIAL AND METHODS: We analyzed retrospectively the data of 282 trauma victims from a non-governmental organizational (NGO) hospital in Sierra Leone, Africa. During a 3-month period (10.10.2015-08.01.2016), these patients had 367 injuries and underwent 263 orthopedic surgeries. Despite a clear indication, some patients did not receive surgical treatment. We identified these injuries and the reason why they could not be operated. The anatomic region of the injury was evaluated and if they had a bone or soft tissue defect or were infected. RESULTS: We identified 95 (25.89%) injuries in 70 patients (47 males; 23 females) that were not be operated. The reasons were lack of specific implants (no implant group; N = 33), no treatment strategy for the injury (no solution group; N = 29), and patients that were lost (lost patient group; N = 33), almost equally distributed by 1/3. In the no implant group were mainly closed fractures and fractures of the pelvis and the proximal femur. The implants needed were locking plates (N = 19), proximal femoral nails (N = 8), and implants for pelvic surgery (N = 6). In the no solution group were nearly all bone (P < 0.0000), soft tissue defects (P < 0.00001) and infections (P = 0.00003) compared to the rest and more open fractures (P < 0.00001). In the lost patients group, most fractures were closed (24 out of 33, P = 0.033). These fractures were mostly not urgent and were postponed repeatedly. CONCLUSION: One quarter of the patients did not receive the surgical treatment needed. Besides acquisition of implants, surgical skills and expertise could be a solution for this issue. Nevertheless, these skills must be passed to local surgeons.

War surgery in Afghanistan: a model for mass causalities in terror attacks?

PURPOSE: The aim of the study was to identify solution strategies from a non-governmental (NGO) hospital in a war region for violence-related injuries and to show how high-income countries (HIC) might benefit from this expertise. METHODS: NGO trauma hospital in Lashkar Gah, Afghanistan. Four hundred eighty-four war victims admitted in a three month period (February 2016-May 2016) were included. Patients´ characteristics were analyzed. RESULTS: The mean age was 23.5 years. Four hundred thirty-four (89.9%) were male, and 50 (10.1%) were female. The most common cause of injury was bullet injuries, shell injuries, and mine injuries. The most common injured body region was the lower extremity, upper extremity, and the chest or the face. Apart from surgical wound care and debridements, which were performed on every wound in the operation theatre, laparotomy was the most common surgical procedure, followed by installation of a chest drainage and amputation. CONCLUSION: The surgical expertise and clear pathways outweigh modern infrastructure. In case of a mass casualty incident, fast decision-making with basic diagnostic means in order to take rapid measurements for life-saving therapies could make the difference.

New Method for Monitoring Partial Weight Bearing (PWB) of Outpatients with an Independent Insole Sensor System.

UNLABELLED: PURPOSE OF THE STUDY Partial weight bearing (PWB) is commonly prescribed post operatively following lower limb fractures and compliance with the weight bearing protocol is an essential element of the rehabilitation. So far it is unknown to what extent patients do comply with PWB during the healing process as instructed by the surgeon. Our aim is to assess a new device for real-time feedback and long-term measurement of PWB of outpatients. The device offers the possibility to monitor the outpatient's activity. The applicability, reliability and validity of the new device should be evaluated. MATERIAL AND METHODS 20 young, healthy subjects complete a course of 500 m that contained several stairs, with a PWB of 15 kg. During the entire test, the axial load, the acceleration and the temperature were measured with a novel insole sensor system. The results were compared with reference measurements performed with a force plate. RESULTS Altogether, the 20 subjects performed 11,106 steps during the completion of the walking circuit. In 23.6% of the steps, the subjects applied a PWB of 10 to 20 kg. In 5.5% of all steps, PWB was superior to 60 kg. The mean bias of the insole was 11,58 N. Limits of agreement were +/- 125 N and the interclass correlation coefficient was r = 0.945. CONCLUSIONS The presented sensor sole might be a useful tool to obtain more precise insight of outpatients' activity and load to the injured limb during the healing process. Furthermore, these results demonstrate that even young and healthy subjects are not able to keep the prescribed PWB. This raises the question, if patients who have been recently operated are able to follow the instructions concerning the PWB. KEY WORDS: partial weight bearing (PWB), insole sensor system, sensor sole, monitoring, outpatients.

[Radiofrequency ablation in spinal osteoid osteoma. Options and limits]. / Radiofrequenzablation bei Osteoidosteomen der Wirbelsäule. Möglichkeiten und Grenzen.

Osteoid osteoma was first described by Jaffe in 1935 as a benign bone neoplasm mainly located in the diaphyseal areas of long bones: 10% are located in the spine, mainly in the lumbar and thoracic posterior elements. Therapy is required due to nocturnal pain independent of the physical load and responds especially well to anti-inflammatory drugs due to the excessive production of prostaglandins in the nidus. Diagnosis is confirmed by multi-slice computed tomography (CT), magnetic resonance imaging (MRI) and skeletal scintigraphy scans. In cases with typical symptoms and imaging, open biopsies are rarely needed. Although CT-guided radiofrequency ablation is accepted as the gold standard treatment option for osteoid osteoma in the extremities, this technique is limited in spinal applications due to the risk of thermal damage to adjacent neurovascular structures. Technical advances in the administration of radiofrequency ablation have, however, resulted in new and expanded indications in the spine so that the necessity for open surgical excision of spinal osteoid osteoma is becoming less.

Molecular mechanisms of DNA damage initiated by alpha, beta-unsaturated carbonyl compounds as criteria for genotoxicity and mutagenicity.

alpha, beta-Unsaturated carbonyl compounds are important not only from a theoretical but also a practical standpoint. These ubiquitous compounds can interact with DNA through various mechanisms. The predominant interaction is the formation of cyclic 1,N2-deoxyguanosine adducts; 7,8-cyclic guanine adducts are also found. We have synthesized and characterized the stereoisomers of adducts formed by about 20 alpha, beta-unsaturated carbonyl compounds. The different types of adducts and the mutagenic and genotoxic response can be explained by the molecular structures of the agents. Compounds forming saturated cyclic adducts are mutagenic in S. typhimurium strain TA100 and to a lesser extent in TA1535. Substances with a leaving group at the C-3 position form unsaturated conjugated cyclic adducts and are mutagenic only in the His D3052 frameshift strains with an intact excision repair system (no urvA mutation). Metabolic epoxidation of the double bond and other metabolic activation, e.g., activation of the nitrogroups via nitroreductases, were also found to contribute to genotoxic and mutagenic activities. Our results have further elucidated the genotoxic mechanisms of these compounds; however, additional investigations are required for a complete understanding of the genotoxic activity of this class of compounds.

Mutagenicity of 2-alkylpropenals in Salmonella typhimurium strain TA 100: structural influences.

alpha,beta-Unsaturated aldehydes are a class of mutagenic and carcinogenic compounds that form promutagenic 1,N(2)-propanodeoxyguanosine adducts. They are important industrial and environmental compounds, are formed endogenously, and are found in food. We recently published structure-mutagenicity relationships for 3-alkyl substituted alpha,beta-unsaturated aldehydes (beta-alkylacroleins) and here we present structural influences on the mutagenicity of the 2-alkyl substituted alpha,beta-unsaturated aldehydes (alpha-alkylacroleins), 2-methylacrolein, 2-ethylacrolein, 2-propylacrolein, and 2-butylacrolein, in Salmonella typhimurium TA 100. All four alkylacroleins are mutagenic without S9-mix; however, the results are strongly influenced by bacterial toxicity of the alkylacroleins. In general, toxicity increases with increasing length of the alkyl substituent. The increasing toxicity with increasing alkyl groups can be explained by increasing lipophilicity that allows the compounds to better penetrate into the bacterial cell. Other structural effects, such as steric hindrance of the deoxyguanosine binding (DNA-adduct formation) and the positive inductive effect of the alkyl groups, have only a slight effect on mutagenesis. Addition of S9-mix leads to an increase in the absolute revertant peak values but a decrease in mutagenic activities, as expressed by revertants per micromol. This effect is also observed with heat-inactivated S9-mix and does not depend on metabolic activation. The effect of S9-mix can be explained by partial detoxication of the substances by nucleophilic components of the S9-mix such as glutathione.

Mutagenicity of beta-alkyl substituted acrolein congeners in the Salmonella typhimurium strain TA100 and genotoxicity testing in the SOS chromotest.

The beta-alkyl substituted acrolein congeners crotonaldehyde, trans-2-pentenal, trans-2-hexenal, 2,4-hexadienal, and trans-2-heptenal were clearly mutagenic in a slightly modified preincubation Ames test with Salmonella typhimurium TA100 with and without S9 mix using a threefold bacterial cell density and a 90-min preincubation time, whereas trans-cis-2,6-nonadienal did not show any mutagenic activity. The greatest impediment to adequate mutagenicity testing of these compounds is their toxicity toward bacteria. Within the congener family tested, toxicity increases as a function of both chain length and lipophilicity, and it becomes more and more difficult to demonstrate mutagenicity. Mutagenicity decreases with increasing chain length. This effect may be explained by increasing toxicity. The effect of S9 mix seems to be mostly nonenzymatic detoxication by nonspecific scavanger protection of bacterial cytotoxicity. No indication could be found that bioactivation plays a role in S9-mediated reduction of bacterial cytotoxicity. Although positive mutagenic outcomes could be obtained with the SOS chromotest for other alpha, beta-unsaturated carbonyl compounds, these acrolein congeners were not genotoxic in this test, most probably because they are toxic for the Escherichia coli bacteria PQ37 and PQ243.

Crotonaldehyde is mutagenic in Salmonella typhimurium TA100.

The mutagenicity of crotonaldehyde in Salmonella typhimurium TA100 cannot be demonstrated in the standard plate incorporation assay. However, as reported earlier by our group, this alpha, beta-unsaturated aldehyde is clearly mutagenic in the liquid assay modification of this testing procedure. Carried out because of the doubts recently expressed by Cooper et al. (Environ Mutagen 9:289-295, 1987) concerning the observed mutagenicity of crotonaldehyde in S. typhimurium TA100, this study confirms that this compound is clearly a direct (without activation by mammalian microsomes)-acting mutagen in S. typhimurium TA100 under appropriate conditions in the preincubation assay. The observed mutagenicity is increased by extended preincubation time and increased bacterial cell densities.

Genotoxicity of 1,3-dichloro-2-propanol in the SOS chromotest and in the Ames test. Elucidation of the genotoxic mechanism.

1,3-Dichloro-2-propanol (1,3-DCP-OH, glycerol dichlorohydrin) is of great importance in many industrial processes and has been detected in foodstuffs, in particular in soup spices and instant soups. It has been shown to be carcinogenic, genotoxic and mutagenic. Its genotoxic mechanisms are, however, not yet entirely understood. We have investigated whether alcohol dehydrogenase (ADH) catalysed activation to the highly mutagenic and carcinogenic 1,3-dichloroacetone or formation of epichlorohydrin or other genotoxic compounds play a role for mutagenicity and genotoxicity. In our studies, no indications of ADH catalysed formation of 1,3-dichloropropane could be found, although we could demonstrate a clear activation by ADH in the case of 2-chloropropenol. Formation of allyl chloride could also be excluded. We found, however, clear evidence that epichlorohydrin formed chemically in the buffer and medium used in the test is responsible for genotoxicity. No indication was found that enzymatic formation of epichlorohydrin plays a role. Additional mutagenicity and genotoxicity studies with epichlorohydrin also confirmed the hypothesis that genotoxic effects of 1,3-DCP-OH depend on the chemical formation of epichlorohydrin.

On the role of alkylating mechanisms, O-alkylation and DNA-repair in genotoxicity and mutagenicity of alkylating methanesulfonates of widely varying structures in bacterial systems.

The Ames test and the SOS-chromotest are widely used bacterial mutagenicity/genotoxicity assays to test potential carcinogens. Though the molecular mechanisms leading to backmutations and to the induction of SOS-repair are in principle known the role of alkylation mechanisms, of different DNA-lesions and of DNA-repair is in parts still unknown. In this study we investigated 14 monofunctional methanesulfonates of widely varying structures for mutagenicity in Salmonella typhimurium strain TA 1535 sensitive for O(6)-guanine alkylation for comparison with strain TA 100 in order to obtain additional information on the role of alkylation mechanisms, formation of the procarcinogenic DNA-lesion O(6)-alkylguanine and the role of DNA-repair in induction of backmutation. The substances were also tested in the SOS-chromotest with Escherichia coli strain PQ 37 and strain PQ 243 lacking alkyl base glycosylases important for base excision repair in order to examine the role of alkylation mechanisms, of base excision repair and the role of O-alkyl and N-alkyl DNA-lesions on the induction of SOS-repair. The secondary methanesulfonates with very high S(N)1-reactivity isopropyl methanesulfonate and 2-butyl methanesulfonate showed highest mutagenicities in both strains. The higher substituted methanesulfonates with very high S(N)1-reactivity had lower mutagenic activities because of reduced half lives due to their high hydrolysis rates. A clear increase in mutagenicities in strain TA 100 was observed for the primary compounds methyl methanesulfonate and allyl methanesulfonate with very high S(N)2-reactivity. The primary compound phenylethyl methanesulfonate has a relatively high mutagenicity in both Salmonella strains which can be explained by an increased S(N)1-reactivity and by low repair of the O(6)-phenylethylguanine. Highest SOSIPs (SOS inducing potency) in strains PQ 37 and PQ 243 were found for methyl methanesulfonate and for the secondary compounds with high S(N)1-reactivity. The ratios in the SOSIPs between strain PQ 243 and PQ 37, indirectly indicative for the role of O- and N-alkylation in the induction of SOS-repair, was high for the primary methanesulfonates and lower for the secondary, indicating that the SOS-repair is, to a certain extent, also induced by other lesions than O(6)-alkylation. The results indicate that O(6)-alkylation is also a predominant lesion for backmutation in strain TA 100 and that in the case of monofunctional alkylating agents high S(N)2-reactivities are required to induce error prone repair mediated backmutations. The O(6)-alkylguanine lesion is also important for induction of SOS-repair in the SOS-chromotest, however, other sites of alkylation which are repaired by the base pair excision repair system can also efficiently contribute to the induction of SOS-repair.

Induction of sfiA SOS function by peroxides using three different E. coli strains.

Five peroxides and two related compounds were tested for genotoxicity by the SOS Chromotest using 3 different E. coli strains (PQ37, PM21, GC4798). All tested hydroperoxides (hydrogen peroxide, tert-butylhydroperoxide, cumene hydroperoxide) were clearly positive in all strains. From a comparison of results obtained from the different strains it can be concluded that neither DNA lesions leading to the induction of excision repair nor covalent binding of radicals to DNA is responsible for the induction of sfiA-SOS function by hydroperoxides. Among the remaining compounds tested, only dibenzoylperoxide gave a clearly positive result in strain PQ37 whereas di-tert-butylperoxide and azobisisobutyronitrile showed only borderline activity. When using strains PM21 and GC4798, none of the latter compounds was positive. Paraquat was inactive in all strains.

The possible role of alpha, beta-unsaturated carbonyl compounds in mutagenesis and carcinogenesis.

alpha, beta-Unsaturated carbonyl compounds are industrially important compounds, ubiquitous in the environment and are formed endogenously. They interact with proteins and enzymes. Genotoxicity was found in eucaryotic cells and some compounds were carcinogenic. Unsaturated carbonyl compounds are considered to play an important role in human cancer. Insufficient and contradictory results were reported on mutagenicity. We demonstrated a clear mutagenic potential for these compounds and have shown interference of their bacterial toxicity with an adequate testing. Structure-mutagenicity relationships were confirmed by the results of the SOS-chromotest. The compounds induce DNA-strand breaks. However, we did not find indications for cross linking. With mutagenic alpha, beta-unsaturated carbonyl compounds we isolated and characterized 1,N2-cyclic deoxyguanosine adducts, 7,8-cyclic and 7-linear guanine adducts as well as 1,N2-7,8-biscyclic adducts and 1,N2-cyclic, 7-linear bisadducts. Reactivity of these compounds towards nucleosides runs in parallel with their mutagenic potential. Mutagenic and carcinogenic activities most probably depend on these reactions with DNA, and DNA adducts can be utilized as indicators for the role of these compounds in human carcinogenicity.

The role of alcohols as solvents in the genotoxicity testing of alpha,beta-unsaturated ketones in the SOS chromotest.

alpha,beta-Unsaturated ketones are bifunctional compounds which form promutagenic 1,N(2)-propanodeoxyguanosine adducts like carcinogenic alpha,beta-unsaturated aldehydes and are mutagenic and genotoxic like these aldehydes. They are important industrial chemicals, are found in our environment and are widespread in our food. We investigated the SOS repair inducing activities of five ketones in the SOS chromotest and compared these results with that of the Ames test. Alkyl substitution at the beta-position of the alpha, beta-unsaturated carbonyl moiety leads to a decrease or loss in genotoxicity. Genotoxicity is higher if using ethanol as solvent instead of dimethylsulfoxide (DMSO). An increasing effect is also observed with methanol and n-propanol. Addition of the alcohol dehydrogenase inhibitor 4-methylpyrazole does not significantly influence the genotoxicity indicating that it is unlikely that the solvent effect depends on competitive inhibition of alcohol dehydrogenase by the alcohols used as solvents. Since other possible explanations e.g. ketal formation or solubility effects are also unlikely, the mechanism of this solvent effect observed with three different E. coli PQ-strains remains unresolved. No significant difference in genotoxicity of ethyl vinyl ketone was found between the strains PQ 37 and PQ 243 indicating that base excision repair does not play a role in the repair of 1,N(2)-propanodeoxyguanosine adducts, the main adducts of the alpha,beta-unsaturated ketones.

Genotoxicity of monofunctional methanesulphonates in the SOS chromotest as a function of alkylation mechanisms. A comparison with the mutagenicity in S. typhimurium TA100.

17 monofunctional methanesulphonates of widely varying structures were investigated in the SOS chromotest using the E. coli strain PQ37. All compounds tested were positive in this assay. The monofunctional methanesulphonates in general possess low SOSiP values. Five of the compounds tested i.e. iBMS, NpMS, 2 PhPMS, PkMS and 1,3-DC12PMS (for abbreviations see Table 1) did not show increasing beta-galactosidase activity and both the positive induction factors and the positive SOSiP values resulted from the toxicity correction as performed according to Quillardet and Hofnung (1985). In general methanesulphonates with a higher SN1 reactivity, in particular the secondary compounds, showed clear genotoxic activities whereas those possessing low SN1 reactivities (primary compounds) induced a low SOS repair indicating that the alkylation of O-atoms in the DNA bases contributes more to the induction of SOS repair in strain PQ37 than N-alkylations. The only exception was methyl methanesulphonate (MMS) which possessed a very high SN2 reactivity but a rather low SN1 reactivity. It had the highest SOSiP value of all tested methanesulphonates. No dependence of the genotoxicity on the SN2 reactivity could be found in this series. In general the phenyl-substituted methanesulphonates showed higher SOSiP values, which is presumably due to their relatively high SN1 reactivities and their relatively long life times in aqueous systems. There is a clear relationship between SN1 reactivities and the SOSiP values: the SOSiP values increase with rising SN1 reactivities reaching a maximum at iPMS after which the genotoxicities decrease due to the decreasing life times. The compounds with very high SN1 reactivities also possess very high hydrolysis rates. A good correlation could be established between the mutagenicities in S. typhimurium TA100 and the SOS chromotest (strain PQ37). Only 4 small deviations from this correlation could be found. The reasons for these deviations are discussed.

Methyl methanesulphonate (MMS) is clearly mutagenic in S. typhimurium strain TA1535; a comparison with strain TA100.

No mutagenicity or an uncertain mutagenic response has been reported in the literature for methyl methanesulphonate (MMS) in S. typhimurium strain TA1535 when using the plate assay. In our studies we found a reproducible mutagenic activity of 62 revertants/mumole and plate for MMS in strain TA1535 when using the preincubation assay. A dose-dependent increase in revertants was, however, observed only at fairly high doses (exceeding 4 mumole). Two different slopes were observed in the dose-response curve when testing MMS with strain TA100. Slope A is dependent on the error-prone response, possible only in strain TA100 due to the pKm101 plasmid (R factor) but not possible in strain TA1535 due to its umuDC deficiency. Slope B observed at higher doses (as in strain TA1535) could be explained through a GC----AT transition initiated by the O6-methylation of guanine. Our findings demonstrate that MMS induces back mutation in S. typhimurium strains carrying the hisG46 missense mutation due to the formation of O6-methylguanine. In the case of strain TA100 the pKm101 plasmid-mediated error-prone mechanism is, however, the predominant process in MMS mutagenesis which leads to a higher mutagenic response at much lower doses than the GT----AT transition in strain TA1535.

Mutagenicity of 2-methylacrolein, 2-ethylacrolein and 2-propylacrolein in Salmonella typhimurium TA100. A comparative study.

The C2-alkylated acrolein derivatives 2-methylacrolein, 2-ethylacrolein and 2-propylacrolein are mutagenic in Salmonella typhimurium TA100. They are direct mutagens, their mutagenic potency being inversely proportional to the size of the alkylating substituent in the C2 position. In the presence of S9 mix, the mutagenicity of all these substances is considerably reduced; the reduction in mutagenicity is inversely proportional to the direct mutagenic potential of the substance. As shown for 2-methylacrolein, the reduction in mutagenicity is dependent on the concentration of S9 in the S9 mix and is not significantly influenced by heat inactivation of the S9 mix or by addition of TCPO, an inhibitor of epoxide hydrolase, to the testing system. There are no indications of enzymatic activation by the metabolizing microsomal system.

Genotoxicity of 2-halosubstituted enals and 2-chloroacrylonitrile in the Ames test and the SOS-chromotest.

2-Chloroacrolein and 2-bromoacrolein are very potent direct mutagens not requiring metabolic activation in Salmonella typhimurium strains TA 100 and TA 1535. Mutagenic activities decrease with increasing degree of methyl substitution at carbon atom C-3 of the acrolein moiety from 2-chloroacrolein via 2-chlorocrotonaldehyde to 2-chloro-3,3-dimethylacrolein. With 2-chloroacrylonitrile equivocal results are obtained in strain TA 100 without S9-mix and unequivocal with S9-mix. In the SOS-chromotest the 2-chloroenals are also very strong genotoxins and the structure-activity relationships found in the Ames test are clearly confirmed. 2-Chloroacrylonitrile is not positive in the SOS-chromotest. The mutagenic mechanisms are discussed, and indications are provided that genotoxicity/mutagenicity depends on formation of DNA adducts, e.g., 1,N2-cyclic deoxyguanosine adducts.

Risk assessment for mutagenic and carcinogenic activities of alpha,beta-unsaturated carbonyl compounds by a screening strategy based on structure-activity relationships.

alpha,beta-Unsaturated compounds are ubiquitous and are formed endogenously. They form DNA adducts and are a constant source of DNA damage. A speedy screening strategy based on structure-activity relationships and a battery of prescreening tests for a rapid and reliable assessment of the role of these compounds in mutagenesis and carcinogenesis is presented and discussed. In this screening strategy, time-consuming and expensive animal tests are replaced by in vitro test with bacteria and cell cultures. The results of the mutagenicity and genotoxicity tests, as well as the results of the binding studies of alpha,beta-unsaturated carbonyl compounds with DNA components, and the corresponding structure-activity relationships, are presented.

The relationship between mutagenicity in His G46 Salmonella and the O(6)-guanine alkylation in bacterial DNA by monofunctional methanesulphonates.

The influence of the O(6)-alkylation in Salmonella typhimurium cells by monofunctional methanesulphonates on their mutagenicity in strains TA1535 and TA100 was examined using a specially modified method that allows DNA-binding studies in bacteria. All the tested monofunctional compounds, methylmethanesulphonate (MMS; very strong Sn2 reactivity), ethylmethanesulphonate (EMS; moderate Sn1 and Sn2 reactivity) and isopropylmethanesulphonate (iPMS; strong Sn1 reactivity) formed significant amounts of O(6)-Palkylguanine. A comparison between the results in a pure in vitro system and the situation in the bacterial cells revealed significant differences. Despite having different alkylating reactivities, MMS and iPMS produced similar amounts of O(6)-adducts in bacterial DNA, probably owing to the much lower hydrolysis rate of MMS when compared with that of iPMS. The O(6):N7 ratio was, however, ten-fold higher in the case of iPMS, which reflects its clearly higher Sn1 reactivity. Owing to the formation of O(6)-adducts, all three test compounds induced back mutations in strain TA1535, which is not capable of performing error-prone repair. Whereas MMS showed a much higher mutagenic activity in strain TA100, because of the large amount of N-alkylation, iPMS induced practically identical mutagenicity in both strains, which indicates that the pkM101 plasmid is not significantly involved in iPMS mutagenicity. Evidently only strong Sn2-type monofunctional alkylating compounds induce back mutation in strain TA100 by the pkM101-mediated error-prone mechanism. This underlines the importance of the O(6)-alkylation as promutagenic lesion in strains containing the His G46 mutation. Surprisingly, EMS showed a higher mutagenicity in strain TA1535 than in TA100.