Biomonitoring of low levels of mercurial derivatives in water and soil by Allium micronucleus assay

The Allium micronucleus (MNC) assay was developed to monitor low levels of mercury in aquatic and terrestrial environments. Four mercurial derivatives namely mercuric chloride (MC), methyl mercuric chloride (MMC), phenyl mercuric acetate (PMA) and a methoxy ethyl mercuric chloride based fungicide, Emisan-6, were tested to assess the sensitivity and versatility of the Allium MNC assay. Allium bulbs were set directly on water and soil contaminated with known levels of mercurial derivatives (0.0001-10.00 ppm). On the 5th day the endpoints measured were root length, mitoses with spindle abnormality and cells with MNC in root meristems. The effective concentrations of the test chemicals that cause 50% of root length as compared to control (EC50) were determined from dose-response curves so obtained. The lowest effective concentration tested (LECT) and highest ineffective concentration tested (HICT) for each of the mercurial derivatives for the induction of spindle malfunction and MNC were determined. It was found that EC50, LECT and HICT values for mercurial derivatives in soil were higher than those in water. The frequencies of cells with MNC and mitoses with spindle abnormality were highly correlated indicating that MNC is a good parameter of spindle malfunction. The present approach increased the sensitivity of the Allium assay by 10-fold, the detection limit being 0.001-0.1 ppm and 0.1-1.0 ppm in aquatic and terrestrial environments respectively, depending on the species of mercury.     

Induction of sister chromatid exchanges by heavy metal salts in root meristem cells ofAllium cepa L.

Four heavy metal salts, nickel sulphate, mercuric chloride, cadmium sulphate and zinc sulphate, were tested for induction of sister chromatid exchange (SCE) in root meristem cells ofAllium cepa. A simple modified Feulgen staining procedure was employed for SCE-analysis. Maleic hydrazide and paraquat were included for comparison. An evaluation of genotoxicity of the above test chemicals made on the basis of SCE-assay was found positive for all the test chemicals with exception of zinc sulphate which gave a weak positive result.     

General method for determining anaerobic biodegradation potential.

A simple, generalized method was refined and validated to test whether an organic chemical was susceptible to anaerobic degradation to CH4 + CO2. The method used digested sewage sludge diluted to 10% and incubated anaerobically in 160-ml serum bottles with 50 micrograms of C per ml of test chemical. Biodegradation was determined by the net increase in gas pressure in bottles with test chemicals over the pressure in nonamended sludge bottles. Gas production was measured by gas chromatography and by a pressure transducer. The latter method is recommended because of its speed, accuracy, and low cost. Sewage sludge from municipal digesters with 15- to 30-day retention times was found to be suitable. The sludge could be stored anaerobically at 4 degrees C for up to 4 weeks with satisfactory test results. p-Cresol, phthalic acid, and ethanol are suggested as reference chemicals to confirm sludge activity and method reliability. A revised anaerobic salts medium was developed which minimizes problems of a biological gas production (CO2), avoids precipitation, and meets the requirements of the anaerobic microbiota. When greater than 75% of the theoretical gas production was observed, the chemical was judged to be degradable, and when 30 to 75% of the expected gas was produced, it was termed partially degradable. This method has been tested on more than 100 chemicals of various physical properties and found to reproducibly determine anaerobic biodegradation potential. Of the chemicals tested, 46 were found to be anaerobically degraded. Sludges from nine different municipal treatment plants were surveyed for their ability to degrade nine chemicals which differed in susceptibility to degradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

General method for determining anaerobic biodegradation potential

A simple, generalized method was refined and validated to test whether an organic chemical was susceptible to anaerobic degradation to CH4 + CO2. The method used digested sewage sludge diluted to 10% and incubated anaerobically in 160-ml serum bottles with 50 micrograms of C per ml of test chemical. Biodegradation was determined by the net increase in gas pressure in bottles with test chemicals over the pressure in nonamended sludge bottles. Gas production was measured by gas chromatography and by a pressure transducer. The latter method is recommended because of its speed, accuracy, and low cost. Sewage sludge from municipal digesters with 15- to 30-day retention times was found to be suitable. The sludge could be stored anaerobically at 4 degrees C for up to 4 weeks with satisfactory test results. p-Cresol, phthalic acid, and ethanol are suggested as reference chemicals to confirm sludge activity and method reliability. A revised anaerobic salts medium was developed which minimizes problems of a biological gas production (CO2), avoids precipitation, and meets the requirements of the anaerobic microbiota. When greater than 75% of the theoretical gas production was observed, the chemical was judged to be degradable, and when 30 to 75% of the expected gas was produced, it was termed partially degradable. This method has been tested on more than 100 chemicals of various physical properties and found to reproducibly determine anaerobic biodegradation potential. Of the chemicals tested, 46 were found to be anaerobically degraded. Sludges from nine different municipal treatment plants were surveyed for their ability to degrade nine chemicals which differed in susceptibility to degradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unscheduled DNA synthesis tests. A report of the U.S. Environmental Protection Agency Gene-Tox Program

The utility of unscheduled DNA synthesis (UDS) testing for screening potentially hazardous chemicals was evaluated using the published papers and technical reports available to the UDS Work Group. A total of 244 documents were reviewed. Based on criteria defined in advance for evaluation of the results, 169 were rejected. From the 75 documents accepted, results were reviewed for 136 chemicals tested using autoradiographic approaches and for 147 chemicals tested using liquid scintillation counting (LSC) procedures; 38 chemicals were tested by both approaches to measure UDS. Since there were no documents available that provided detailed recommendations of UDS screening protocols or criteria for evaluating the results, the UDS Work Group presents suggested protocols and evaluation criteria suitable for measuring and evaluating UDS by autoradiography in primary rat hepatocytes and diploid human fibroblasts and by the LSC approach in diploid human fibroblasts. UDS detection is an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs, because it measures the repair of DNA damage induced by many classes of chemicals over the entire mammalian genome. However, for this system to be utilized effectively, appropriate metabolic activation systems for autoradiographic measurements of UDS in human diploid fibroblasts must be developed, the nature of hepatocyte-to-hepatocyte variability in UDS responses must be determined, and the three suggested protocols must be thoroughly evaluated by using them to test a large number of coded chemicals of known in vivo mutagenicity and carcinogenicity.     

The improved Allium/Vicia root tip micronucleus assay for clastogenicity of environmental pollutants

The meristematic mitotic cells of plant roots are appropriate and efficient cytogenetic materials for the detection of clastogenicity of environmental pollutants, especially for in situ monitoring of water contaminants. Among several cytological endpoints in these fast dividing cells, such as chromosome/chromatid aberrations, sister-chromatid exchanges and micronuclei, the most effective and simplest indicator of cytological damage is micronucleus formation. Although the Allium cepa and Vicia faba root meristem micronucleus assays (Allium/Vicia root MCN) have been used in clastogenicity studies about 12 times by various authors in the last 25 years, there is no report on the comparison of the efficiency of these two plant systems and in different cell populations (meristem and F₁) of the root tip as well as under adequate recovery duration. In order to maximize the efficiency of these bioassays, the current study was designed to compare the Allium and the Vicia root MCN assays on the basis if chromosome length, peak sensitivity of the mitotic cells, and the regions of the root tip where the MCN are formed. The total length of the 2n complement of Allium chromosomes is 14.4 μm and the total length of the 2n complement of Vicia is 9.32 μm. The peak sensitivity determined by serial fixation at 12-h intervals after 100 R of X-irradiation is 44 h. The slope of the X-ray dose-response curve of Allium roots derived from the meristematic regions was lower than that derived from cells in the F₁ region. Higher efficiency was also demonstrated when the MCN frequencies were scored from the F₁ cells in both Allium and Vicia treated with formaldehyde (FA), mitonycin C (MMC), and maleic hydrazide (MH). The results indicated that scoring of MCN frequencies from the F₁ cell region of the root tip was more efficient than scoring from the meristematic region. The X-ray linear regression dose-response curves were established in both Allium and Vicia cell systems and the coefficients of correlations, slope values were used to verify the reliability and efficiency of these two plant cell systems. Based on the dose-response slope value of 0.894 for Allium and 0.643 for Vicia, the Allium root MCN was a more efficient test system. The greater sensitivity of the Allium roots is probably due to the greater total length of the diploid complement and the higher number of metacentric chromosomes. The Allium/Vicia root MCN test system was applied to determine the clastogenicity of saccharin (SC) and wastewater from Rio Queretaro and the Arena canal in the city of Queretaro, Mexico. The minimum effective dose (MED) is 10 R for X-rays, 50 mM for FA, 2.2 mM for MMC, 0.01 mM for MH, and 40 ppm for SC.     

Optimum associations of tester strains for maximum detection of mutagenic compounds in the Ames test

The Ames test is now widely used as a short-term test for the detection of mutagens. Different strains are available with various genetic characteristics, and in the past decade various authors have recommended different associations of strains to give maximum detection potential. However, few studies have been done to compare the sensitivity of individual strains towards a wide range of compounds in a single study. In order to define the best association of strains for screening or regulatory purpose, we have tested 103 direct mutagens (reference genotoxins or in-house compounds) on 7 strains of Salmonella typhimurium: TA1535, TA1537, TA1538, TA97, TA98, TA100 and TA102. 126 different associations of strains have been studied in terms of sensitivity and percentage overlap. Optimum associations of 2, 3, 4 or 5 strains included strains both with and without plasmid pKM101. However, the specificity of detection is greatly diminished by the presence of plasmid pKM101 in the strain, as shown by the high degree of overlap in associations constituted entirely of strains containing the plasmid. The association of strains TA1538 and TA100 detected 86% of the chemicals tested and is therefore recommended for large-scale screening. A rate of detection of 100% was obtained when 6 strains were used. The best associations of 4 and 5 strains, which detected 97 and 99% chemicals respectively, all contained strains TA1537, TA1538 and TA102. Finally, the associations of 4 strains (TA1537, TA1538, TA100, TA102) or 5 strains (TA1535, TA1537, TA1538, TA97, TA102) seemed well adapted to the optimum detection of mutagenic compounds.     

The present status of higher plant bioassays for the detection of environmental mutagens

Higher plants provide valuable genetic assay systems for screening and monitoring environmental pollutants. They are now recognized as excellent indicators of cytogenetic and mutagenic effects of environmental chemicals and are applicable for the detection of environmental mutagens both indoor and outdoor. Comparisons between plant and nonplant genetic assay systems indicate that higher plant genetic assays have a high sensitivity (i.e. few false negatives). Two assays which are considered ideal for in situ monitoring and testing of airborne and aqueous mutagenic agents are the Tradescantia stamen hair assay for mutations and the Tradescantia micronucleus assay for chromosome aberrations. Both assays can be used for in vivo and in vitro testing. Other higher plant gentoxicity assys which have a large number of genetic markers and/or data base and are also highly suitable for testing for genotoxic agents include Arabidopsis thaliana, Allium cepa, Hordeum vulgare, Vicia faba, and Zea mays. Since higher plant systems are now recognized as excellent indicators of the cytotoxic, cytogenetic, and mutagenic effects of environmental chemicals and have unique advantages for in situ monitoring and screening it is recommended that higher plant systems be accepted by regulatory authorities as an alternative first-tier assay system for the detection of possible genetic damage resulting from pollution or the use of environmental chemicals. The results from higher platn genetic assays could meke a significant contribution in protecting the public from agents that can cause mutation and cancer. The advantages possessed by higher plant genetic assays, which are inexpensive and easy to handle, make them ideal for use by scientists in developing countries.     

Genotoxicity of maleic hydrazide,acridine and DEHP in Allium cepa root cells performed by two different laboratories

The purpose of this paper was to compare the results of the Allium cepa chromosome aberration assay between two laboratories under the same test protocol and at the same time, use chemicals and onions obtained in their own homeland. For this study three chemicals were selected: di(2-ethylhexyl)phthalate (DEHP), maleic hydrazide, and acridine. Both laboratories found genotoxicity with a positive dose-response relationship for maleic hydrazide and acridine. However, for DEHP the results were quite different--one of the laboratories found this compound not genotoxic but the other found a positive response. Although the comparative study was inconclusive for DEHP, it was successful for the maleic hydrazide, acridine and also for the positive control (methyl methanesulfonate). Further studies need to be performed in the case of DEPH.     

Saccharomyces cerevisiae BLYAS, a new bioluminescent bioreporter for detection of androgenic compounds

A Saccharomyces cerevisiae strain, capable of autonomous bioluminescence, was engineered to respond to androgenic chemicals. The strain, S. cerevisiae BLYAS, contains the human androgen receptor in the chromosome and was constructed by inserting a series of androgen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 that constitutively expressed luxA and luxB to create pUTK420. Cotransformation of this plasmid with a second plasmid (pUTK404), containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp), yielded a bioluminescent bioreporter responsive to androgenic chemicals. Using dihydrotestosterone (DHT) as a standard, the response time and the 50% effective concentration values were 3 to 4 h and (9.7 +/- 4.6) x 10(-9) M, respectively. The lower limit of detection in response to DHT was 2.5 x 10(-9) M, and in response to testosterone it was 2.5 x 10(-10) M. This strain is suitable for high-throughput screening of chemicals with potential for remote environmental monitoring systems because of the assay speed, sensitivity, and self-containment.     

Xenoestrogenic compounds promote capacitation and an acrosome reaction in porcine sperm

There is growing evidence that endocrine disruptors bind to hormone receptors; since these receptors are present on the sperm membrane, sperm are potentially a useful model for examining estrogenic activities of endocrine disruptors. The objective of the present study was to compare the effects of two xenoestrogenic compounds (genistein and 4-tert-octylphenol) to those of two steroids (estrogen and progesterone) and heparin on in vitro capacitation and the acrosome reaction in a porcine sperm model. Porcine sperm were incubated with various concentrations (0.001-100 μM) of each chemical for 15 or 30 min, and then capacitation and the acrosome reaction were assessed using chlortetracycline. Estrogen and progesterone were considerably more potent than the other chemicals in stimulating capacitation. Estrogen stimulated sperm capacitation at all tested concentrations after 15 min of incubation (P < 0.05), whereas progesterone stimulated sperm capacitation at all tested concentrations after 15 and 30 min (P < 0.05). The effect of genistein on sperm capacitation was comparable with that of estrogen, and it was the most potent in stimulating the acrosome reaction. Genistein stimulated the acrosome reaction at all tested concentrations after 30 min (P < 0.05). However, 4-tert-octylphenol had the least effect on capacitation and the acrosome reaction. In summary, since all chemicals studied effectively altered capacitation and the acrosome reaction, it was concluded that porcine sperm could be a useful model for in vitro screening of potential endocrine disruptors. It was noteworthy that concurrent comparisons to steroids increased the ability to determine estrogenic characteristics of the tested chemicals.     

The EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritation tests--an assessment of the performance of the optimised test

During the past decade, several validation studies have been conducted on in vitro methods for discriminating between skin irritating and non-irritating chemicals. The reconstructed human skin models, EpiDerm and EPISKIN, provided the most promising results. Based on experience of the similar performance of the two skin models, it was suggested that a common test protocol and prediction model should be developed for the prediction of skin irritation potential with the two models. When the EPISKIN protocol was applied with the EpiDerm model, an acceptable specificity (80%) was achieved, whereas the sensitivity (60%) was low. In 2003, the EPISKIN protocol was further refined by extending the post-incubation period following exposure to test chemicals. This extension and additional technical improvements to the EpiDerm protocol were evaluated with 19 chemicals from the prevalidation study. With the new test design, high sensitivity (80%) and specificity (78%) were obtained. The statistical probability for correct classifications was high, so the test was considered to be ready for formal validation. However, since test optimisation had been conducted with the same test chemicals as were used in the ECVAM prevalidation study, it was decided that the optimisation of the protocol had to be verified with a new set of chemicals. Thus, in the current study, 26 additional chemicals (10 rabbit irritants and 16 non-irritants), which had previously been selected and tested by LOREAL with EPISKIN, were evaluated in three independent experiments with EpiDerm. With this unbalanced testing set, a specificity of 94%, and a sensitivity of 60% were obtained, while the positive and negative predictivity and accuracy remained almost unchanged (around 80%) in comparison to the in vivo rabbit data. Overall, 45 chemicals (20 irritants and 25 non-irritants) were tested according to the final protocol. The resulting high positive (82%) and negative predictive values (79%) confirmed the reliability (accuracy of 80%) of the improved test protocol of the EpiDerm model.     

A system for assessment of chemical substances on mutagenicity in man: general priniciples, practical recommendations and further elaboration]

As a basis of the suggested test-system, the following conditions are observed: 1) the economy of fulfilment in a short time; 2) the analysis of gene and chromosome mutations in germ and somatic cells; 3) the evaluation of mutagenic effects of not only substance, but also of the products of its metabolism; 4) including in the system only the tests which give the minimal variability between separate experiments; 5) the evaluation of dose-effect relationship. The practical scheme of testing is divided into two parts: a screening and a complete one. The screening programme consists of two tests: a) a test on microorganisms with a metabolic activation in vitro; b) a cytogenetic analysis of bone marrow of mammals. The complete programme of testing includes 4 tests: a) a test on microorganisms with a metabolic activation in vitro and in vivo; b) a test of dominant lethal mutations on mammals; c) a cytogenetic analysis of bone marrow of mammals; d) a cytogenetic analysis in the culture of human lymphocytes. There are good reasons for the principles of selection of substance for testing according to the screening and complete programme: population occurence, economic (or medical) significance, information about relative chemicals showing mutagenic, carcinogenic and teratogenic effect. In the group of chemicals which are to be tested according to the screening programme, such ones can be included: industrial chemicals, phosphoorganic insecticides, drugs which are taken by a limited group of patients. The group of chemicals which are to be tested according to the complete programme consists of the following ones: pesticides, food additices, widespread drugs, the chemicals of the group 1, if during one of the tests of the screening programme a genetic effect is detected. At the genetic risk estimation it is advisable to keep to the following rule: a positive effect, identified in any object of the system must in the direct meaning extrapolate on men. The quantitative evaluation of the mutagenic danger of a chemical can be determined by the increase of the spontaneous level of mutations in the test-object on the basis of an average dose and exposition of the given chemical in the human population. Those chemicals are subject to the quantitative evaluation, which have shown a mutagenic activity during any of the test-objects; they are also widespread and because of their social or economic value can not be replaced or excluded from taking. From the point of view of genetics any substance with a mutagenic activity is dangerous and must be prohibited from using or replaced by any other non-mutagenic chemical, or limited by the contact of persons of non-reproductive age. As a temporary measure from a hygienic point of view, it is recommended to evaluate this chemical as especially mutagenic and prohibit or limit its using, when its average population dose produces 1/10 or more increase of the spontaneous level of mutations.     

Validation of the rat limb bud micromass test in the international ECVAM validation study on three in vitro embryotoxicity tests

A detailed report is presented on the performance of the rat limb bud micromass (MM) test in a European Centre for the Evaluation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects on animals and/or humans, were tested in four laboratories. The outcome showed that the MM test is an experimentally validated test, which holds promise for use for identifying strongly embryotoxic chemicals, but which needs to be improved before it can be recommended for use for regulatory purposes.     

Allium test for air and water borne pollution control

An Allium cepa test was used to study the genotoxicity of the ecosystem. Elevated frequencies of chromosomal aberrations (CA) and root growth inhibition were observed in Allium as a result of exposure to genotoxicants investigated in rainfall, snowfall, a petrochemical polluted site and in aquarium water, compared with the relevant control. The Allium test has been shown to be a useful tool for the detection of potentially genotoxic substances in air and water screening programmes.     

Cytotoxic and genotoxic effects of dioxacarb by human peripheral blood lymphocytes CAs and Allium test

Dioxacarb (Elecron, Famid) is a phenyl methylcarbamate insecticide and in vitro cytotoxic and genotoxic effects of this pesticide on human peripheral blood lymphocytes and Allium root meristematic cells were investigated by chromosomal aberrations (CAs) and Allium test. Human lymphocytes were treated with 62.5, 125, 250 and 500 ppm doses of dioxacarb for CAs. CA/cell, abnormal cell % and mitotic index % (MI %) data were obtained from these concentrations in 24 and 48 h treatment periods. Dioxacarb did not increase the CA/cell frequency significantly, so this insecticide was not identified as genotoxic. But it was found cytotoxic especially at 250 and 500 ppm concentrations because of the reduced the MI % and increased the abnormal cell %. In Allium test, 25 ppm (EC50/2), 50 ppm (EC50) and 100 ppm (EC50 × 2) concentrations were used for root growth inhibition (EC50 determination) and Allium mitotic index (MI) determination tests. The used concentrations of dioxacarb induced dose-dependent inhibition of MI and root growth on root meristems. Mitotic inhibition of dioxacarb was found significantly higher than for the positive control. These Allium results indicated the high cytotoxicity of dioxacarb. The present study is the first research on cytotoxicity and genotoxicity of dioxacarb by human lymphocyte CAs and Allium test.     

Immunotoxic and cytotoxic effects of atrazine, permethrin and piperonyl butoxide to rainbow trout following in vitro exposure

For many current use pesticides, limited information exists on their cytotoxicity and immunotoxicity in non-target organisms such as fish. We examined the effects of atrazine, permethrin and piperonyl butoxide (PBO) exposure, in vitro, on rainbow trout (Oncorhynchus mykiss) lymphocyte viability and proliferation. Purified rainbow trout peripheral blood leukocytes (PBLs) were exposed in vitro to the test chemicals (0, 0.01, 0.1, 1 and 10 μM) for 96 h, with and without the mitogen lipopolysaccharide. All three chemicals caused a decrease in both lymphocyte viability and proliferation at 10 μM, while atrazine also suppressed proliferation of PBLs at 1 μM. The in vitro toxicity of these chemicals to this salmonid underscores the need for further investigation using in vivo studies and host resistance models.     

Large-scale virtual screening for the identification of new Helicobacter pylori urease inhibitor scaffolds

Here, we report a structure-based virtual screening of the ZINC database (containing about five million compounds) by computational docking and the analysis of docking energy calculations followed by in vitro screening against H. pylori urease enzyme. One of the compounds selected showed urease inhibition in the low micromolar range. Barbituric acid and compounds 1a, 1d, 1e, 1f, 1g, 1h were found to be more potent urease inhibitors than the standard inhibitor hydroxyurea, yielding IC(50) values of 41.6, 83.3, 66.6, 50, 58.8, and 60 μM, respectively (IC(50) of hydroxyurea = 100 μM). 5-Benzylidene barbituric acid has enhanced biological activities compared to barbituric acid. Furthermore, the results indicated that among the substituted 5-benzylidene barbiturates, those with para substitution have higher urease inhibitor activities. This may be because the barbituric acid moiety is closer to the bimetallic nickel center in unsubstituted or para-substituted than in ortho- or meta-substituted analogs, so it has greater chelating ability.     

Impact of reduced vancomycin susceptibility on the therapeutic outcome of MRSA bloodstream infections

Background

Resistance to anti-tuberculosis drugs is a serious public health problem. Multi-drug resistant tuberculosis (MDR-TB), defined as resistance to at least rifampicin and isoniazid, has been reported in all regions of the world. Current phenotypic methods of assessing drug susceptibility of M. tuberculosis are slow. Rapid molecular methods to detect resistance to rifampicin have been developed but they are not affordable in some high prevalence countries such as those in sub Saharan Africa. A simple multi-well plate assay using mycobacteriophage D29 has been developed to test M. tuberculosis isolates for resistance to rifampicin. The purpose of this study was to investigate the performance of this technology in Kampala, Uganda.

Methods

In a blinded study 149 M. tuberculosis isolates were tested for resistance to rifampicin by the phage assay and results compared to those from routine phenotypic testing in BACTEC 460. Three concentrations of drug were used 2, 4 and 10 μg/ml. Isolates found resistant by either assay were subjected to sequence analysis of a 81 bp fragment of the rpoB gene to identify mutations predictive of resistance. Four isolates with discrepant phage and BACTEC results were tested in a second phenotypic assay to determine minimal inhibitory concentrations.

Results

Initial analysis suggested a sensitivity and specificity of 100% and 96.5% respectively for the phage assay used at 4 and 10 μg/ml when compared to the BACTEC 460. However, further analysis revealed 4 false negative results from the BACTEC 460 and the phage assay proved the more sensitive and specific of the two tests. Of the 39 isolates found resistant by the phage assay 38 (97.4%) were found to have mutations predictive of resistance in the 81 bp region of the rpoB gene. When used at 2 μg/ml false resistant results were observed from the phage assay. The cost of reagents for testing each isolate was estimated to be 1.3US$ when testing a batch of 20 isolates on a single 96 well plate. Results were obtained in 48 hours.

Conclusion

The phage assay can be used for screening of isolates for resistance to rifampicin, with high sensitivity and specificity in Uganda. The test may be useful in poorly resourced laboratories as a rapid screen to differentiate between rifampicin susceptible and potential MDR-TB cases.     

In vivo and in vitro cytogenetic effects of the anti-tumor agent amsacrina (AMSA)

The genotoxic effect of AMSA, an anti-tumor agent, was evaluated using the micronucleus and anaphase-telophase tests. The doses assayed by the in vivo micronucleus test were 1.5, 3 and 6 mg/kg: they are within the range of those used in clinical trials. A significant increase of micronucleated cells (P less than 0.01) was observed in the three assayed doses, with a linear dose response (r 0.98). In the in vitro test, 3 drug concentrations, i.e. 10, 1 and 0.1 microgram/ml, were analyzed with the 2 higher doses. AMSA showed a marked inhibition of cellular replication, but with 0.1 microgram/ml it was possible to determine an increase (P less than 0.01) in aberrations in anaphase-telophase cells. Both studies clearly demonstrate the clastogenic effect of the drug, which should be taken into account when considering its carcinogenic risk.