Exogenous Metabolic System Research Articles

Reconstructed human intestinal comet assay, a possible alternative in vitro model for genotoxicity assessment.

The aim of the present study was to evaluate the compatibility of reconstructed 3D human small intestinal microtissues to perform the in vitro comet assay. The comet assay is a common follow-up genotoxicity test to confirm or supplement other genotoxicity data. Technically, it can be performed utilizing a range of in vitro and in vivo assay systems. Here, we have developed a new reconstructed human intestinal comet (RICom) assay protocol for the assessment of orally ingested materials. The human intestine is a major site of food digestion and adsorption, first-pass metabolism as well as an early site of toxicant first contact and thus is a key site for evaluation. Reconstructed intestinal tissues were dosed with eight test chemicals: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), phenformin hydrochloride (Phen HCl), benzo[a]pyrene (BaP), 1,2-dimethylhydrazine hydrochloride (DMH), potassium bromate (KBr), glycidamide (GA), and etoposide (Etop) over a span of 48 h. The RICom assay correctly identified the genotoxicity of EMS, ENU, KBr, and GA. Phen HCl, a known non-genotoxin, did not induce DNA damage in the 3D reconstructed intestinal tissues whilst showing high cytotoxicity as assessed by the assay. The 3D reconstructed intestinal tissues possess sufficient metabolic competency for the successful detection of genotoxicity elicited by BaP, without the use of an exogenous metabolic system. In contrast, DMH, a chemical that requires liver metabolism to exert genotoxicity, did not induce detectable DNA damage in the 3D reconstructed intestinal tissue system. The genotoxicity of Etop, which is dependent on cellular proliferation, was also undetectable. These results suggest the RICom assay protocol is a promising tool for further investigation and safety assessment of novel ingested materials. We recommend that further work will broaden the scope of the 3D reconstructed intestinal tissue comet assay and facilitate broader analyses of genotoxic compounds having more varied modes of actions.

Abstract 5338: A novel in vitro assay to evaluate the roles of hepatic metabolism on anticancer drug safety and efficacy

Abstract It has been well-established that hepatic drug metabolism may have significant impact on the efficacy and off-target toxicity of anticancer drugs. We present here an in vitro assay, the metabolism-dependent cytotoxicity assay (MDCA), for the evaluation of the roles of specific pathways on the efficacy of anticancer drugs. The assay employs a novel in vitro hepatic metabolic system, the permeabilized cofactor-supplemented human hepatocytes (MetMax Human Hepatocytes, MMHH), as an exogenous metabolic activating system for the evaluation of the in vitro cytotoxicity of anticancer agents towards a designated tumor and nontumor cells. MMHH are derived from cryopreserved human hepatocytes isolated from human livers procured for but not used for transplantation provided to our laboratory from the International Institute for the Advancement of Medicine. We have previously demonstrated that MMHH retained all drug metabolizing enzyme pathways present in human hepatocytes. The advantages of MMHH include the following: 1. Drug metabolizing enzyme activities are not affected by cytotoxicity of the drug substrate, a major limitation of intact human hepatocytes; and 2. Drug metabolism pathways can be selected by cofactor specification (e.g. NADPH/NAD+ for phase 1 oxidation; Uridine 5′-diphosphoglucuronic acid (UDPGA) for glucuronidation; 3'-phosphoadenosine 5'-phosphosulfate (PAPS) for sulfation; N-acetyl coenzyme A for N-acetylation). We present here a proof-of-concept study to evaluate the role of various hepatic metabolic pathways towards the cytotoxicity of cyclophosphamide, a commonly used drug for the treatment of renal carcinoma, towards a renal cancer cell line, HEK293 cells. We observed that NADPH enhanced the cytotoxicity, consistent with the known metabolic activation of cyclophosphamide by P450 isoforms (e.g. CYP2B6; CYP3A4) to the known metabolic activation of cyclophosphamide to 4-hydroxycloophosphamide, followed by the formation of the ultimate cytotoxic metabolites phosphoramide mustard and acrolein, which are responsible for its anticancer properties. In addition, we found that the addition of L-glutathione attenuated cyclophosphamide cytotoxicity towards the HEK293 cells, an observation consistent with the roles of this cellular detoxification system in the resistance of tumor cells to chemotherapeutic agents. Minimal effects were observed for UDPGA and PAPS, consistent with the known noninvolvement of these phase 2 conjugating pathways in cyclophosphamide metabolism. MDCA represents an in vitro experimental system that can be routinely applied towards the discovery and development of novel anticancer drugs to evaluate the roles of drug metabolism in efficacy and safety, with various cancer cells (e.g. prostate carcinoma, neuroblastoma) and cells from normal tissues (e.g. cardiomyocytes, intestinal mucosal cells), respectively, as target cells. Citation Format: Albert P. Li, Hong Wei. A novel in vitro assay to evaluate the roles of hepatic metabolism on anticancer drug safety and efficacy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5338.

Permeabilized Cryopreserved Human Hepatocytes as an Exogenous Metabolic System in a Novel Metabolism-Dependent Cytotoxicity Assay for the Evaluation of Metabolic Activation and Detoxification of Drugs Associated with Drug-Induced Liver Injuries: Results with Acetaminophen, Amiodarone, Cyclophosphamide, Ketoconazole, Nefazodone, and Troglitazone.

We report here a novel in vitro experimental system, the metabolism-dependent cytotoxicity assay (MDCA), for the definition of the roles of hepatic drug metabolism in toxicity. MDCA employs permeabilized cofactor-supplemented cryopreserved human hepatocytes (MetMax Human Hepatocytes, MMHH), as an exogenous metabolic activating system, and human embryonic kidney 293 (HEK293) cells, a cell line devoid of drug-metabolizing enzyme activity, as target cells for the quantification of drug toxicity. The assay was performed in the presence and absence of cofactors for key drug metabolism pathways known to play key roles in drug toxicity: NADPH/NAD+ for phase 1 oxidation, uridine 5'-diphosphoglucuronic acid (UDPGA) for uridine 5'-diphospho-glucuronosyltransferase (UGT) mediated glucuronidation, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for cytosolic sulfotransferase (SULT) mediated sulfation, and glutathione (GSH) for glutathione S-transferase (GST) mediated GSH conjugation. Six drugs with clinically significant hepatoxicity, resulting in liver failure or a need for liver transplantation: acetaminophen, amiodarone, cyclophosphamide, ketoconazole, nefazodone, and troglitazone were evaluated. All six drugs exhibited cytotoxicity enhancement by NADPH/NAD+, suggesting metabolic activation via phase 1 oxidation. Attenuation of cytotoxicity by UDPGA was observed for acetaminophen, ketoconazole, and troglitazone, by PAPS for acetaminophen, ketoconazole, and troglitazone, and by GSH for all six drugs. Our results suggest that MDCA can be applied toward the elucidation of metabolic activation and detoxification pathways, providing information that can be applied in drug development to guide structure optimization to reduce toxicity and to aid the assessment of metabolism-based risk factors for drug toxicity. GSH detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites, a key property of drugs with idiosyncratic hepatotoxicity. SIGNIFICANCE STATEMENT: Application of the metabolism-dependent cytotoxicity assay (MDCA) for the elucidation of the roles of metabolic activation and detoxification pathways in drug toxicity may provide information to guide structure optimization in drug development to reduce hepatotoxic potential and to aid the assessment of metabolism-based risk factors. Glutathione (GSH) detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites that may be applied toward the evaluation of idiosyncratic hepatotoxicity.

HepGentox: a novel promising HepG2 reportergene-assay for the detection of genotoxic substances in complex mixtures.

BackgroundIn risk assessment, genotoxicity is a key factor to determine the safety for the consumer. Most in vitro genotoxicity assays were developed for the assessment of pure substances. However, in recent years more attention has been given to complex mixtures, where usually low amounts of a substance are present. For high-throughput screening, a toxicologically sensitive assay should be used, covering a broad range of genotoxic substances and detecting them at low concentrations. HepG2 cells have been recommended as one of the prime candidates for genotoxicity testing, as they are p53 competent, less prone towards cytotoxic effects and tend to have some metabolic activity.MethodsA HepG2 liver cell line was characterized for its suitability for genotoxicity assessment. For this, a luciferase based reporter gene assay revolving around the p53 pathway was validated for the analysis of pure substances and of complex mixtures. Further, the cell’s capability to detect genotoxins correctly with and without an exogenous metabolizing system, namely rat liver S9, was assessed.ResultsThe assay proved to have a high toxicological sensitivity (87.5%) and specificity (94%). Further, the endogenous metabolizing system of the HepG2 cells was able to detect some genotoxins, which are known to depend on an enzymatic system. When complex mixtures were added this did not lead to any adverse effects concerning the assays performance and cytotoxicity was not an issue.DiscussionThe HepGentox proved to have a high toxicological sensitivity and specificity for the tested substances, with similar or even lower lowest effective concentration (LEC) values, compared to other regulatory mammalian assays. This combines some important aspects in one test system, while also being less time and material consuming and covering several genotoxicity endpoints. As the assay performs well with and without an exogenous metabolizing system, no animal liver fractions have to be used, which application is discussed controversially and is considered to be expensive and laborious in sample testing. Because of this, the HepGentox is suitable for a cost-efficient first screening approach to obtain important information with human cells for further approaches, with a relatively fast and easy method. Therefore, the HepGentox is a promising assay to detect genotoxic substances correctly in complex mixtures even at low concentrations, with the potential for a high throughput application. In a nutshell, as part of an in vitro bioassay test battery, this assay could provide valuable information for complex mixtures.

MetMax Human Hepatocyte/HEK Cytotoxic Reactive Metabolite Assay as a Potential In Vitro Experimental System for the Identification of DILI Drugs

In drug development, it is important to identify drug candidates where drug metabolism plays key roles in their toxicity. Major reasons for the identification of drug candidates with metabolism-dependent toxicity are as follows: 1. Safety profiles determined in preclinical animal species may not accurately predict human effects due to species difference in drug metabolism; and 2. idiosyncratic drug toxicity may occur in individuals due to enhanced metabolic activation or compromised detoxification. We recently developed a novel in vitro hepatocyte system, the MetMax Human Hepatocyte (MMHH) system with permeabilized human hepatocytes supplemented with metabolic cofactors, for the evaluation of drug metabolism. The permeabilization treatment removes the plasma membrane as a barrier for drug diffusion into the hepatocytes, and the diffusion of the metabolites of the hepatocytes. A further advantage of MMHH is that cytotoxicity of the intended substrate would not affect drug metabolism, a challenge often encountered in the evaluation of metabolism of toxic drugs in intact hepatocytes. We report here the development of the MMHH/HEK assay for the evaluation of metabolic activation and detoxification of toxic drugs. In this assay, drug toxicity is quantified in a drug metabolism incompetent target cell line (HEK293) in the presence of absence of MMHH as an exogenous metabolic activating system, and in the presence of selective cofactors for the evaluation of key metabolic activation and detoxification pathways. As a proof-of-concept study, the MMHH/HEK assay was applied in the the evaluation of the model metabolically-activated drugs that are known to be associated with severe drug induced liver injuries (DILI) - acetaminophen, cyclophosphamide, and troglitazone. Our results showed that the cytotoxicity of these model DILI drugs was enhanced in the presence of MMHH supplemented with NADPH, thereby confirming the role of oxidative metabolism in metabolic activation. Furthermore, supplementation of NADPH with reduced glutathione (GSH), a physiologically relevant detoxifying conjugating cofactor for reactive metabolites, led to significant attenuation of cytotoxicity, demonstrating that these model drugs were metabolized by hepatocytes to to reactive, cytotoxic metabolites. Supplementation with UDPGA and PAPS also attenuated the in vitro cytotoxicity of these model drugs in the MMHH/HEK assay, demonstrating the involvement of glucuronidation and sulfation conjugating pathways in toxification. Troglitazone cytotoxicity in the MMHH/HEK assay was found to be higher than that observed for the known less hepatotoxic analogs, rosiglitazone and pioglitazone. Our results suggest that MMHH/HEK assay can be applied towards the evaluation of metabolic activation and detoxification and the identification of drugs with idiosyncratic DILI potential.

In vitro biochemical characterization and genotoxicity assessment of Sapindus saponaria seed extract

Ethnopharmacological relevanceSapindus saponaria, also popularly known as soapberry, has been used in folk medicinal values because of its therapeutic properties and several compounds in its composition, which represent a target in potential for drug discovery. However, few data about its potential toxicity has been reported. Aim of the studyPlant proteins can perform essential roles in survival, acting as defense mechanism, as well functioning as important molecular reserves for its natural metabolism. The aim of the current study was to investigate the in vitro toxicity profile of protein extract of S. saponaria and detect protein potentially involved in biological effects such as collagen hydrolysis and inhibition of viral proteases. Materials and methodsProtein extract of soapberry seeds was investigated for its cytotoxic and genotoxic action using the Ames test. The protein extract was also subjected to a partial purification process of a protease and a protease inhibitor by gel chromatography filtration techniques and the partially isolated proteins were characterized biochemically. ResultsSeed proteins extract of S. saponaria was evaluated until 100 μg/mL concentration, presenting cytotoxicity and mutagenicity in bacterial model mostly when exposed to exogenous metabolic system and causing cytotoxic and genotoxic effects in HepG2 cells. The purification and partial characterization of a serine protease (43 kDa) and a cysteine protease inhibitor (32.8 kDa) from protein extract of S. Saponaria, corroborate the idea of ​​the biological use of the plant as an insecticide and larvicide. Although it shows cytotoxic, mutagenic and genotoxic effects. ConclusionThe overall results of the present study provide supportive data on the potential use of proteins produced in S. saponaria seeds as pharmacological and biotechnological agents that can be further explored for the development of new drugs.

Performance of HepaRG and HepG2 cells in the high-throughput micronucleus assay for in vitro genotoxicity assessment

ABSTRACT The micronucleus (MN) assay is a core test used to evaluate genotoxic potential of xenobiotics. The traditional in vitro MN assay is usually conducted in cells lacking metabolic competency or by supplementing cultures with an exogenous rat S9 metabolic system, which creates a significant assay limitation for detecting genotoxic metabolites. Our previous study demonstrated that compared to HepG2, HepaRG cells exhibited a significantly higher level of CYP450 enzyme activities and detected a greater portion of genotoxic carcinogens requiring metabolic activation using the Comet assay. The aim of this study was to assess the performance of HepaRG cells in the flow cytometry-based MN assay by testing 28 compounds with known genotoxic or carcinogenic modes of action (MoA). HepaRG cells exhibited higher sensitivity (83%) than HepG2 cells (67%) in detecting 12 indirect-acting genotoxicants or carcinogens. The HepaRG MN assay was 100% specific and 93% accurate in detecting genotoxic potential of the 28 compounds. Quantitative comparison of the MN concentration-response data using benchmark dose analysis showed that most of the tested compounds induced higher % MN in HepaRG than HepG2 cells. In addition, HepaRG cells were compatible with the Multiflow DNA damage assay, which predicts the genotoxic MoA of compounds tested. These results suggest that high-throughput flow cytometry-based MN assay may be adapted using HepaRG cells for genotoxicity assessment, and that HepaRG cells appear to be more sensitive than HepG2 cells in detecting genotoxicants or carcinogens that require metabolic activation.

HPLC-ESI-QqQ based standardization, mutagenic and genotoxic potential of methanol extract of Ziziphus mauritiana Lam leaves.

The leaves of Ziziphus mauritiana Lam have been an integral part of the traditional system of medicine for the treatment of inflammation, wounds, fever, asthma and liver disorders. The leaves are utilised as an edible vegetable in rural parts of India and Indonesia. Despite its pharmacological significance, Ziziphus mauritiana Lam lacks scientific evidence on its mutagenic and genotoxic potential. The aim of the present work is to identify bioactive compounds present in the methanol extract of Ziziphus mauritiana Lam leaves (MEZ) using HPLC-ESI-QqQ and to evaluate its mutagenic and genotoxic potential. The phytochemical standardization of the MEZ was done using HPLC-ESI-QqQ. The mutagenic and genotoxic potential of MEZ was tested using bacterial reverse mutation (Ames test), chromosomal aberration, and micronucleus tests. The Ames test was performed in Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537, and the genotoxic potential was tested in in-vitro using chromosome aberration assay with Chinese hamster ovary (CHO) cells and in-vivo micronucleus test using mouse bone marrow cells. Fifteen phytochemical compounds were identified in HPLC-ESI- QqQ. It was observed from the Ames test that MEZ did not induce gene mutations in the S. typhimurium in the presence or absence of S9 activation. Similarly, no significant increase in the number of structural aberrations was observed in CHO cells with or without S9 activation. The oral administration of MEZ at a dose of up to 2000 mg/kg caused no significant increase in the number of micronucleated polychromatic erythrocytes or in the mean ratio of polychromatic to total erythrocytes. The findings of the present study confirm that MEZ is not-mutagenic and non-genotoxic in the presence or absence of the exogenous metabolizing system.

Chloroethene. Documentation of proposed values of occupational exposure limits (OELs)

Chloroethene. Documentation of proposed values of occupational exposure limits (OELs)

Identification of biotransformation products of disperse dyes with rat liver microsomes by LC-MS/MS and theoretical studies with DNA: Structure-mutagenicity relationship using Salmonella/microsome assay

Azo dyes are known as a group of substances with DNA damage potential that depend on the nature and number of azo groups connected to aromatic rings (benzene and naphthalene), chemical properties, e.g. solubility and reactive functional groups, which significantly affect their toxicological and ecological risks. In this paper, we used in vitro models to evaluate the metabolism of selected textile dyes: Disperse Red 73 (DR 73), Disperse Red 78 (DR 78) and Disperse Red 167 (DR 167). To evaluate the mutagenic potential of the textile dyes, the Salmonella mutagenicity assay (Ames test) with strains TA 98 and TA 100 in the presence and absence of the exogenous metabolic system (S9) was used. DR73 was considered the most mutagenic compound, inducing both replacement base pairs (TA 100) and also changing frameshift (TA 98) mutations that are reduced in the presence of the S9 mixture. Furthermore, we used rat liver microsomes in the same experimental conditions of the S9 mixture to metabolize the dyes and the resultant solutions were analyzed using a liquid chromatography coupled to a quadrupole linear ion trap mass spectrometry (LC-MS/MS) to investigate the metabolites formed by the in vitro biotransformation. Based on this experiment, we detected and identified two biotransformation products for each textile dye substrate analyzed. Furthermore, to evaluate the interaction and reactivity of these compounds with DNA, theoretical calculations were also carried out. The results showed that the chemical reaction occurred preferentially at the azo group and the nitro group, indicating that there was a reduction in these groups by the CYP P450 enzymes presented in the rat microsomal medium. Our results clearly demonstrated that the reduction of these dyes by biological systems is a great environmental concern due to increased genotoxicity for the body of living beings, especially for humans.

The influence of exogenous metabolism on the specificity of in vitro mammalian genotoxicity tests.

A two-part study was designed to determine whether the inclusion of the rodent liver 'S9' exogenous metabolic activating system contributes to the generation of misleading positive results by the regulator-required in vitro mammalian genotoxicity tests. The mono-oxygenase enzymes in S9 produce direct-acting DNA-reactive electrophiles, and are included in in vitro genotoxicity tests to enhance the detection of substances which only become genotoxic following metabolism. However, as the S9 system lacks 'detoxifying' phase 2 factors it was hypothesised that increased chemical metabolism per se may lead to an increase in irrelevant S9 test outcomes in safety assessment. To test this, 89 compounds with positive or negative carcinogenicity data were identified, which produced negative Ames test data (+/- S9), and only produced positive in vitro mammalian test data in the presence of S9. This allowed a determination of whether or not misleading predictions of carcinogenicity by the in vitro mammalian tests were more or less prevalent in the presence of S9. A subset of these compounds was then tested with and without S9 in the GADD45a-GFP genotoxicity test, in order to determine whether misleading in vitro mammalian positive results were generally more prevalent with S9, or reflected particular tests' liabilities. This study suggests that the use of S9 metabolic activation in in vitro genotoxicity tests does not increase the prevalence of misleading positive results in in vitro mammalian genotoxicity assays, at least amongst Ames negative compounds.

Toxicogenetic effects of low concentrations of the pesticides imidacloprid and sulfentrazone individually and in combination in in vitro tests with HepG2 cells and Salmonella typhimurium

The insecticide imidacloprid and the herbicide sulfentrazone are two different classes of pesticides that are used for pest control in sugarcane agriculture. To evaluate the genotoxic potential of low concentrations of these two pesticides alone and in mixture, the comet assay and the micronucleus (MN) test employing fluorescence in situ hybridization (FISH) with a centromeric probe were applied in human hepatoma cell lines (HepG2), in a 24-h assay. Mutagenicity was assessed by Salmonella/microsome assay with TA98 and TA100 strains in the absence and presence of an exogenous metabolizing system (S9). The results showed significant inductions of MN in HepG2 cells by both pesticides, for all the tested concentrations. As evidenced in the comet assay, only the imidacloprid presented significant responses. When the two pesticides were associated, a significant induction of damage was observed in the HepG2 cells by the comet assay, but not by the MN test. Moreover, the MN induced by the mixtures of the pesticides appeared at lower levels than those induced by sulfentrazone and imidacloprid when tested alone. According to the FISH results, the damage induced by imidacloprid in the HepG2 cells resulted from a clastogenic action of this insecticide (76.6% of the MN did not present a centromeric signal). For the herbicide sulfentrazone and for the mixture of the pesticides, a similar frequency of MN with and without the presence of the centromeric signal (herbicide: 52.45% of the MN without centromeric signal and 47.54% of the MN with centromeric signal; mixture: 48.71% of the MN without centromeric signal and 51.42% of the MN with centromeric signal) was verified. Based on these results, it was concluded that each one of the pesticides evaluated interacts with the DNA of HepG2 cells and causes irreparable alterations in the cells. However, the combination of the pesticides showed an antagonistic effect on the cells and the damage induced was milder and not persistent in HepG2 cells. The results obtained by the Ames test did not point out significant results.

Cytological assays for the evaluation of genotoxicity in plants: A review

Plants model are recognized as excellent genetic models to detect environ-mental mutagens and are frequently used in xenomoitoring studies. Several assays such as chromosomal aberration assay, micronucleus assay and comet assay are used for detecting the mutagenicity, cytotoxicity and genotoxicity of environmental pollutants. It is easily handled and has advantages over other short-term tests that require pervious preparation of tested samples as well as the addition of exogenous metabolic system. Plants model also enables to evaluate different end points such as chromosomal aberration assay (CAA), micronucleus assay (MNA) and comet assay (CA) that have been used to detect genotoxicity of environmental pollutants. In addition, plants model provides important information to evaluate action mechanisms of an agent about its effects on the genetic material (clastogenic and/or aneugenic effects). It has been widely used to assess the impacts caused by xenobiotic, characterizing an important tool for environmental monitoring studies. The present review describes the two important assays using plant models that are appropriate and efficient cytogenetic materials for the detection of geno-toxicity of environmental pollutants.

Assessment of mutagenic, antimutagenic and genotoxicity effects of Mimosa tenuiflora

Genotoxic effects of Mimosa tenuifl ora (Willd.) Poir, Fabaceae, were investigated by using both micronucleus test and bacterial reverse mutation assay in Salmonella typhimurium TA97, TA98, TA100, TA102 respectively. In respect of Ames test results show that the extract does not induce mutations in any strains of Salmonella typhimurium tested since the mutagenicity index is less than 2. In the antimutagenic effect was observed that the extract at the concentrations tested signifi cantly decreased the mutagenicity index of all strains tested which characterized the extract as antimutagenic in these conditions. In the micronucleus test in vivo, we observed that the concentrations used did not induce an increase in the frequency of micronucleus in normochromatic erythrocytes of mice. Therefore, we concluded that the extract of M. tenuiflora is not mutagenic in the absence of exogenous metabolizing system and does not induce an increase in the frequency of the micronucleus characterized as an agent not mutagenic in these conditions. Further studies of toxicity need to be made to the use of this plant in the treatment of diseases to be stimulated.

Preliminary Study by Environmental Indicator Measurements of Sediments in a Mangrove Forest in Ilha Grande Bay, Rio de Janeiro, Southeastern Brazil

Mangroves perform essential functions in biological cycles and are protected by law in Brazil. However, they are being degraded above their support limit, leading to a reduction in the areas around the world. The area investigated in the present study is a potentially unpolluted mangrove ecosystem in the south of Rio de Janeiro state, Brazil. The aim of this work was to investigate the presence of metals and polycyclic aromatic hydrocarbons (PAHs) in mangrove sediment and to correlate them with mutagenic and genotoxic activity. Four seasonal samplings undertaken between 2009 and 2010 at five sites were analyzed. Dichloromethane extracts were obtained under sonication and subjected to mutagenicity assays using Salmonella microsuspension. Metal (Cd, Cr, Cu, Ni and Pb) contents were determined by atomic absorption spectrometry, while the quantification of PAHs was performed by gas chromatography-mass spectrometry. Goniopsis cruentata hemocytes were used to assess potential genotoxic damage (Micronucleus assay). The absence of mutagenicity was observed for all samples using Salmonella typhimurium strains TA97, TA98, TA100 and TA102, in the absence and presence of an exogenous mammalian metabolizing system. Among the metals, Cd and Cu were detected in autumn at values exceeding the threshold effect level and below the probable effect level. Benzo[a] pyrene was detected at levels above the threshold at one site, where small effects could be observed in the biota. Significant differences in the micronucleated cells observed suggest DNA damage had been induced by the PAHs identified.

A battery of genotoxicity studies with an allergy vaccine adjuvanted with monophosphoryl lipid A (MPL®) for the treatment of grass pollen allergy

The allergy vaccine Pollinex® Quattro Grass, developed for the prevention/relief of allergic symptoms caused by grass pollen in adults and children, contains extracts of 12 grass pollens and rye cereal (all chemically modified by glutaraldehyde), adsorbed onto L-tyrosine with addition of the immunostimulatory adjuvant monophosphoryl lipid A (MPL®). This paper represents the first publication on genetic toxicology data for such a vaccine. An Ames test using five strains of Salmonella typhimurium at concentrations up to 2000 standardized units (SU) per plate in the absence (-) and presence (+) of metabolic activation (rat liver S9) showed negative results, as did treatment - S9 in the mouse lymphoma assay (MLA). However, a reproducible positive response was noted in the MLA + S9, which could not be attributed to extreme culture conditions, but may have been an artefact of the exogenous metabolizing system. Up to 60% false positive results are reported for the MLA with noncarcinogens. Hence, in vivo genotoxicity studies were conducted. These further assessments comprised a rat bone marrow micronucleus test following subcutaneous (s.c.) doses of 10 000, 20 000 or 40 000 SU kg⁻¹ per day for 2 days, and a comet assay in liver and kidney cells from rats treated with Pollinex® Quattro Grass at 20 000 or 40 000 SU kg⁻¹ per day s.c. for 2 days. Plasma analysis showed evidence of systemic antibodies to Pollinex® Quattro Grass immunogens, but the exposure levels could not be quantified. No evidence of genotoxicity was observed in either of the in vivo studies. Overall, the genotoxicity test results supported safe clinical use of Pollinex® Quattro Grass.

Allium cepa test in environmental monitoring: A review on its application

Higher plants are recognized as excellent genetic models to detect environmental mutagens and are frequently used in monitoring studies. Among the plant species, Alium cepa has been used to evaluate DNA damages, such as chromosome aberrations and disturbances in the mitotic cycle. Employing the A. cepa as a test system to detect mutagens dates back to the 40s. It has been used to this day to assess a great number of chemical agents, which contributes to its increasing application in environmental monitoring. The A. cepa is characterized as a low cost test. It is easily handled and has advantages over other short-term tests that require previous preparations of tested samples, as well as the addition of exogenous metabolic system. Higher plants, even showing low concentrations of oxidase enzymes and a limitation in the substrate specification in relation to other organism groups, present consistent results that may serve as a warning to other biological systems, since the target is DNA, common to all organisms. The A. cepa test also enables the evaluation of different endpoints. Among the endpoints, chromosome aberrations have been the most used one to detect genotoxicity along the years. The mitotic index and some nuclear abnormalities are used to evaluate citotoxicity and analyze micronucleus to verify mutagenicity of different chemicals. Moreover, the A. cepa test system provides important information to evaluate action mechanisms of an agent about its effects on the genetic material (clastogenic and/or aneugenic effects). In the face of all the advantages that the A. cepa test system offers, it has been widely used to assess the impacts caused by xenobiotics, characterizing an important tool for environmental monitoring studies, where satisfactory results have been reported.

Using the Salmonella assay to delineate the dispersion routes of mutagenic compounds from coal wastes in contaminated soil

The mutagenicity of acidic and organic extracts of surface soil under the influence of a coal-fired power plant was evaluated by Salmonella/microsome assay using strains TA97a, TA98 and TA100 in the absence and presence of exogenous metabolic systems (S9 mix). Additionally, strains YG1041 and YG1042 (sensitive to nitroderivatives) were used for the organic extracts. In general, the responses were higher in the organic extracts in the presence of S9 mix. The comparison between strains TA98 and TA100 and their derived strains YG1041 and YG1042, respectively, allowed the detection of the presence of nitro-aromatic compounds in some sampling areas, which was confirmed by chemical analysis. The interpretation of the set of mutagenesis data suggests that there are two important mutagenic compound dispersion routes in the area of study: frameshift mutagens were dispersed predominantly by runoff and leaching, while base-pair substitution mutagens were dispersed mainly by the atmosphere. This mutagenic damage might be attributed to the effects of several substances detected in the area, such as aliphatic hydrocarbons and the metals aluminum, cadmium, lead and iron.

Organic-solvent extraction of model biomaterials for use in thein vitro chromosome aberration test

We prepared polyurethane (PU) containing 0.4% or 4% 4,4'-methylenedianiline (MDA) as model materials to investigate the effectiveness of sample preparation by organic-solvent extraction for the in vitro chromosome aberration (CA) test. MDA itself (0.4 mg/mL) was positive only in the presence of an exogenous metabolizing system (S9 mix). The culture medium extract of PU containing 4% MDA (PU/4% MDA) was negative with and without S9 mix. Methanol and acetone extracts, on the other hand, induced structural CAs without S9 mix, which we did not expect because MDA requires S9 mix for activity. On chemical analysis, however, we found that the ratio of MDA extracted by the organic solvents to that extracted by the culture medium of PU/4% MDA was about 15:1. Interestingly, oligomers consisting of poly(tetramethyleneglycol) derivatives (OTMG) were also extracted by the organic solvents. The data suggest that the induction of structural CAs in the absence of S9 mix may have been partly due to synergism of MDA and OTMG. CA tests of MDA and PTMG-1000 in combination confirmed that to be the case. Thus, organic-solvent extraction may be more effective than medium extraction in evaluating the biological safety of biomaterials. Detailed chemical analysis of extracts was performed.

Sediment Mutagenicity and Liver Oxidative Stress in Fish Under Tannery Influence

The mutagenicity of interstitial water and organic extracts from the sediments in the Cadeia and Feitoria Rivers, RS, Brazil, were evaluated by Salmonella microsuspension bioassay using TA97a, TA98, TA100 and TA102 strains, in the absence and presence of S9 mix. At the contaminated site, the mutagenic responses for interstitial water, suggested the presence of frameshift and base pair substitution mutagens, including substances that generate oxidative stress. Organic extracts presented direct or indicative mutagenesis to the TA97a, TA98 and TA100 strains. In general, an exogenous metabolic systems decreased the mutagenicity of the samples. The livers of Gymnogeophagus gymnogenys, family Cichlidae, collected in this impacted area, compared to a non-polluted site, were analyzed for oxidative stress parameters. Compared to the controls, there was a significant increase in the activity of superoxide dismutase (SOD), levels of substances reactive to thiobarbituric acid (TBARS), and in the chemiluminescence of hepatic cells in fish collected at the polluted area. The concentration of cytochromes P450 and b5 decreased drastically in the fish at the polluted site, while the catalase activity did not change. It was possible to correlate the biological changes in the fish with the presence of mutagenic compounds in interstitial water in this area.