Effects of long-term exposure to 50 Hz magnetic fields on cell viability, genetic damage, and sensitivity to mutagen-induced damage.

Until today, it remains controversial whether long-term exposure to extremely low-frequency magnetic fields (ELF-MF) below the legislative exposure limits could result in adverse human health effects. In the present study, the effects of long-term in vitro MF exposure on three different study endpoints (cell viability, genetic damage, and sensitivity to damage induced by known mutagens) were investigated in the human B lymphoblastoid (TK6) cell line. Cells were exposed to 50 Hz MF at three selected magnetic flux densities (i.e., 10, 100, and 500 µT) for different exposure periods ranging from 96h up to 6 weeks. Cell viability following MF exposure was assessed using the ATP-based cell viability assay. Effects of MF exposure on cell genetic damage and cell sensitivity to mutagen-induced damage were evaluated using the in vitro alkaline comet assay and the in vitro cytokinesis block micronucleus assay. The results showed that long-term exposure up to 96h to 50 Hz MF at all tested flux densities could significantly increase TK6 cell viability. In contrast, long-term MF exposure did not affect cell genetic damage, and long-term pre-exposure to MF did not change cell sensitivity to damage induced by known mutagens. At certain time points, statistically significant difference in genotoxicity test results were observed between the MF-exposed cells and the control cells. However, these observations could not be confirmed in the repeat experiments, indicating that they are probably not biologically significant.

Impact of experimental design factors on the potency of genotoxicants in in vitro tests.

Previous studies have shown that differences in experimental design factors may alter the potency of genotoxic compounds in in vitro genotoxicity tests. Most of these studies used traditional statistical methods based on the lowest observed genotoxic effect levels, whereas more appropriate methods, such as the benchmark dose (BMD) approach, are now available to compare genotoxic potencies under different test conditions. We therefore investigated the influence of two parameters, i.e. cell type and exposure duration, on the potencies of two known genotoxicants [aflatoxin B1 and ethyl methanesulfonate (EMS)] in the in vitro micronucleus (MN) assay and comet assay (CA). Both compounds were tested in the two assays using two cell types (i.e. CHO-K1 and TK6 cells). To evaluate the effect of exposure duration, the genotoxicity of EMS was assessed after 3 and 24 h of exposure. Results were analyzed using the BMD covariate approach, also referred to as BMD potency ranking, and the outcome was compared with that of more traditional statistical methods based on lowest observed genotoxic effect levels. When comparing the in vitro MN results obtained in both cell lines with the BMD covariate approach, a difference in potency was detected only when EMS exposures were conducted for 24 h, with TK6 cells being more sensitive. No difference was observed in the potency of both EMS and aflatoxin B1 in the in vitro CA using both cell lines. In contrast, EMS was more potent after 24 h exposure compared with a 3 h exposure under all tested conditions, i.e. in the in vitro MN assay and CA in both cell lines. Importantly, for several of the investigated factors, the BMD covariate method could not be used to confirm the differences in potencies detected with the traditional statistical methods, thus highlighting the need to evaluate the impact of experimental design factors with adequate approaches.

Ecotoxicity profile of heavily contaminated surface water of two rivers in Tunisia.

Persistent organic and inorganic contaminants generated by industrial effluent wastes poses a threat to the maintenance of aquatic ecosystems and public health. The Khniss and Hamdoun rivers, located in the central-east of Tunisia, receive regularly domestic and textile wastewater load. The present study aimed to survey the water quality of these rivers using physicochemical, analytical and toxicological approaches. In the physicochemical analysis, the recorded levels of COD and TSS in both samples exceed the Tunisian standards. Using the analytical approach, several metals and some textile dyes were detected. Indeed, 17 metals were detected in both samples in varying concentrations, which do not exceed the Tunisian standards. The sources of metals pollution can be of natural and anthropogenic origin. Three textile disperse dyes were detected with high levels compared to other studies: the disperse orange 37 was detected in the Khniss river with a concentration of 6.438 µg/L and the disperse red 1 and the disperse yellow 3 were detected in the Hamdoun river with concentrations of 3.873 µg/L and 1895 µg/L, respectively. Textile activities were the major sources of disperse dyes. For both samples, acute and chronic ecotoxicity was observed in all the studied organisms, however, no genotoxic activity was detected. The presence of metals and textile disperse dyes could be associated with the ecotoxicological effects observed in the river waters, in particular due to the industrial activity, a fact that could deteriorate the ecosystem and therefore threaten the human health of the population living in the study areas. Combining chemical and biological approaches, allowed the detection of water ecotoxicity in testing organisms and the identification of possible contributors to the toxicity observed in these multi-stressed water reservoirs.

Identification of flavouring substances of genotoxic concern present in e-cigarette refills.

E-cigarettes have become very popular, a trend that has been stimulated by the wide variety of available e-liquid flavours. Considering the large number of e-liquid flavours (>7000), there is an urgent need to establish a screening strategy to prioritize the flavouring substances of highest concern for human health. In the present study, a prioritization strategy combining analytical screening, in silico tools and literature data was developed to identify potentially genotoxic e-liquid flavourings. Based on the analysis of 129 e-liquids collected on the Belgian market, 60 flavourings with positive in silico predictions for genotoxicity were identified. By using literature data, genotoxicity was excluded for 33 of them whereas for 5, i.e. estragole, safrole, 2-furylmethylketon, 2,5-dimethyl-4-hydroxyl-3(2H)-furanone and transhexanal, there was a clear concern for in vivo genotoxicity. A selection of 4 out of the remaining 22 flavourings was tested in two in vitro genotoxicity assays. Three out of the four tested flavourings induced gene mutations and chromosome damage in vitro, whereas equivocal results were obtained for the fourth compound. Thus, although there is a legislative framework which excludes the use of CMR compounds in e-liquids, flavourings of genotoxic concern are present and might pose a health risk for e-cigarette users.

In vitro 50 Hz magnetic field long-term exposure: Cytogenetic tests on human lymphoblastoid TK6 cells and validation of the test environment.

Potential health effects of extremely low-frequency (electro)magnetic fields (ELF-(E)MFs) have long been investigated, but the results are still inconclusive. With respect to genotoxicity, sound data related to the effects of long-term exposure to ELF-(E)MFs on the genetic material and the impact of long-term pre-exposure to ELF-(E)MFs on the sensitivity of cells to the damage induced by known mutagens are needed. In this manuscript, an optimized protocol for a combined in vitro comet/micronucleus study to investigate these effects in a human lymphoblastoid cell line (TK6) is provided including the description of a well-validated exposure system. Furthermore, the use of a shielding system to limit background ELF-MFs inside the incubator is described as well.•Optimized protocols for cytogenetic tests with ELF-MFs on TK6 cells ensure the reproducibility of test results.•Validation of exposure environment and exposure system are needed prior to performing tests with ELF-MFs.•A simple, but effective method to shield cells and reduce unintentional ELF-MF exposure consists of using the mu-metal cylinder. This is of particular interest when studying the effects of low exposure levels.

Assessment of natural coagulants to remediate Tunisian textile wastewater by combining physicochemical, analytical, and toxicological data.

Due to the complexity and variability of textile wastewater composition, a constant search for new treatment strategies that are efficient, eco-friendly, and cost-effective is mandatory. In the present study, the efficiency of coagulation-flocculation using biocoagulants derived from cactus Opuntia ficus indica and eggplant Solanum melongena to remove toxic compounds from Tunisian textile wastewater samples was evaluated by combining assays to investigate physicochemical properties and in vitro (geno)toxicity with analytical chemistry. Both natural coagulants could significantly improve the physicochemical properties of the textile wastewater samples compared to the traditionally used chemical coagulant. The highest rate of decolorization was achieved after treatment with the cactus-derived coagulant. The analytical study revealed the presence of only crystal violet dye (CV) in only one sample. Both natural coagulants were able to remove CV, which may (partially) explain the decolorization of the treated samples. Only one untreated textile effluent induced a genotoxic response in the VITOTOX® assay. The genotoxic effect was not linked to the presence of CV and was no longer observed after treatment with each of the natural coagulants, suggesting the effectiveness of the remediation treatments to remove potentially genotoxic compound(s). However, in the other genotoxicity tests, no biologically relevant effects were observed for any of the tested samples. In conclusion, although the physicochemical data indicate that the use of natural coagulants (cactus and eggplant) could be an interesting alternative treatment process to the chemical coagulant for detoxifying textile effluents, these results were only partially supported by the toxicological and analytical data.

UPLC-MS/MS analysis of antibiotics in pharmaceutical effluent in Tunisia: ecotoxicological impact and multi-resistant bacteria dissemination.

The UPLC MS/MS analysis showed the presence of the two antibiotics in the pharmaceutical industry discharges during 3 months; norfloxacin and spiramycin which were quantified with the mean concentrations of 226.7 and 84.2 ng mL-1, respectively. Sixteen resistant isolates were obtained from the pharmaceutical effluent and identified by sequencing. These isolates belong to different genera, namely Citrobacter, Acinetobacter, Pseudomonas, Delftia, Shewanella, and Rheinheimera. The antibiotic resistance phenotypes of these isolates were determined (27 tested antibiotics-discs). All the studied isolates were found resistant to amoxicillin and gentamicin, and 83.33% of isolates were resistant to ciprofloxacin. Multiple antibiotic resistances were revealed against ß-lactams, quinolones, and aminoglycosides families. Our overall results suggest that the obtained bacterial isolates may constitute potential candidates for bioremediation and can be useful for biotechnological applications. Genotoxic effects were assessed by a battery of biotests; the pharmaceutical wastewater was genotoxic according to the bacterial Vitotox test and micronuclei test. Genotoxicity was also evaluated by the comet test; the tail DNA damages reached 38 and 22% for concentrated sample (10×) and non-concentrated sample (1×), respectively. However, the histological sections of kidney and liver's mice treated by pharmaceutical effluent showed normal histology and no visible structural effects or alterations as cytolysis, edema, or ulcerative necrosis were observed. Residual antibiotics can reach water environment through wastewater and provoke dissemination of the antibiotics resistance and induce genotoxic effects.

Identification and risk assessment of human and veterinary antibiotics in the wastewater treatment plants and the adjacent sea in Tunisia.

In the following study, we came up with and validated a prompt, sensitive and precise method for the simultaneous determination of 56 antimicrobial drugs (tetracyclines, sulfonamides, ß-lactams, macrolides and quinolones) using the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This method was implemented with success to determine antibiotics in samples collected from four wastewater treatment plants and five coasts in Tunisia. Results showed the presence of high concentrations of antibiotics that ranged from 0.1 to 646 ng mL-1, which can induce many negative impacts on health and the environment. For this reason, we have opted to evaluate toxicity of wastewater samples using a battery of biotests. In fact, genotoxicity was assessed using three tests: Vitotox, comet and micronucleus assays. The input and output of wastewater treatment plants induced a strongly genotoxic effect on the Salmonella typhimurium TA104 prokaryotic Vitotox assay. This result was confirmed using the comet and the micronucleus assays performed on the human liver hepatocellular carcinoma cells. The genotoxic power of the tested wastewater treatment plants' samples could be attributed to the presence of the higher quantities of antibiotics that are detected in these samples and to the antibiotic and organic compound cocktails.

Antigenotoxic prenylated flavonoids from stem bark of Erythrina latissima.

A series of prenylated flavonoids was obtained from antigenotoxic extracts and fractions of stem bark of Erythrina latissima E. Mey (Leguminosae). In addition to five constituents never reported before, i.e. (2S)-5,7-dihydroxy-2-(4-hydroxy-2-(prop-1-en-2-yl)-2,3-dihydrobenzofuran-6-yl)chroman-4-one (erylatissin D), (2S)-5,7-dihydroxy-2-(4-methoxy-2-(prop-1-en-2-yl)-2,3-dihydrobenzofuran-6-yl)chroman-4-one (erylatissin E), 5,7-dihydroxy-3-(4-methoxy-2-(prop-1-en-2-yl)-2,3-dihydrobenzofuran-6-yl)-4H-chromen-4-one (erylatissin F), (2S)-5,7,8'-trihydroxy-2',2'-dimethyl-[2,6'-bichroman]-4-one (erylatissin G) and (2S)-5,7-dihydroxy-8'-methoxy-2',2'-dimethyl-[2,6'-bichroman]-4-one (dihydroabyssinin I), 18 known flavonoids were identified. Evaluation of the antigenotoxic properties (against genotoxicity induced by aflatoxin B1, metabolically activated) in the Vitotox assay revealed that most flavonoids were active. Sigmoidin A and B showed the highest activity, with an IC50 value of 18.7 µg/mL, equivalent to that of curcumin (IC50 18.4 µg/mL), used as a reference antigenotoxic compound.

In vitro Toxicity and Genotoxic Activity of Aqueous Leaf Extracts From Four Varieties of Olea europea (L).

AIM: Despite its therapeutic value almost nothing is known about potential adverse health effects of Olea europea L. We therefore investigated the in vitro toxicity and genotoxicity of leaf extracts of this plant. MATERIAL AND METHODS: Extracts from olive tree leaves were obtained from four different regions in Tunisia. We investigated the in vitro toxicity, genotoxicity and antigenotoxicity of their aqueous extracts using the neutral red (NR) uptake, Vitotox and alkaline comet assays. RESULTS: None of the extracts were found to be toxic and none of them were genotoxic, although some doubt exists for the extract obtained at Meski (North of Tunisia). On the basis of the Vitotox test only, none of the extracts appeared to have antigenotoxic (or cogenotoxic) properties. DISCUSSION: The negative genotoxicity underline the safe use of the leaves, for example, as hypoglycemic and antidiabetic preparations. Lack of antigenotoxicity may indicate that the previously reported anticancer effects do not result from protection against genotoxicity. HIGHLIGHTS: We investigated the in vitro toxicity and genotoxicity of aqueous extracts of olivesThe neutral Red Uptake test, Vitotox and alkaline comet assay were usedLeaf extracts from 4 different origins were investigatedNone of them showed in vitro toxicity or genotoxicityThe extracts also didn't have antigenotoxic properties Abbreviation list: BaP: benzo(α)pyrene, EMS: ethyl methane sulfonate, LMP: low melting point, NI50: 50% inhibition of NRU, NR: neutral red, NRU: neutral red uptake, OD: optical density, PBS: phosphate buffer saline, SDS: sodium dodecyl sulphate, S/N: signal to noise ratio, 4NQO: 4-nitroquinoline oxide.

Evaluation of the Genotoxicity and Cytotoxicity of Semipurified Fractions from the Mediterranean Brown Algae, Dictyopteris membranacea.

Dictyopteris membranacea, a species of Mediterranean brown algae, is believed to have potential pharmacological and nutritional applications. However, such potentials only make sense when devoid of any adverse health consequences. The present study should be seen in this context. It aimed at evaluating the genotoxicity and cytoxicity of its organic extract (F0) and semi purified fractions (F4, F5, and F6). Extracts were tested using the bacterial Vitotox® test and micronucleus assay in different concentrations (from 1.25 µg/mL up to 100 µg/mL, depending on the test and the extract). Applied concentrations were based on a preliminary dose-finding test with the neutral red uptake assay. The results show that all extracts were not genotoxic in the presence or absence of a rat metabolic enzyme fraction (S9). This is encouraging and justifies further investigations on the therapeutic and other values of this algae. SUMMARY: Dictyopteris membranacea extracts and some of their semi purified fractions have important antibacterial properties.The organic extract (F0) and semi purified fractions (F4, F5, and F6) were not genotoxic according to the bacterial Vitotox test.They were also not genotoxic according to the micronucleus test in human C3A cells.Applied concentrations were based on the in-vitro neutral red uptake (NRU) test.

The Cytome Assay as a Tool to Investigate the Possible Association Between Exposure to Extremely Low Frequency Magnetic Fields and an Increased Risk for Alzheimer's Disease.

Exposure to extremely low frequency magnetic fields (ELF-MF) has been identified as one of the potential environmental risk factors for Alzheimer's disease (AD). However, this is far from being established. So far there is no experimental evidence supporting this alleged association. We have performed an in vitro cytogenetic laboratory investigation to explore the plausibility of such association. Our investigation was based on possible similarities found in cells from AD patients and in cells exposed to ELF-MF. We especially found that 50  Hz ELF-MF increase the frequency of cells with (large) micronuclei and nuclear buds indicating that fields above 50 µT may induce chromosome instabilities as those found in AD patients. It should be stressed yet that results from the few published experimental studies on ELF-MF and AD are rather reassuring. Thus, our findings certainly do not prove anything. They only suggest that further investigations might be necessary.

Co-exposure of ELF-magnetic fields and chemical mutagens: An investigation of genotoxicity with the SOS-based VITOTOX test in Salmonella typhimurium.

It is believed that extreme low frequency magnetic fields (ELF-MF) are not mutagenic, at least at exposure levels below 100 µT. Synergistic or co-operative effects with environmental mutagens remain possible yet. We therefore investigated the effects of ELF-MF in conjunction with 4 different well known chemical mutagens having different modes of action. For this purpose the bacterial Vitotox test was used. Our study confirmed previous results which showed that a 100 µT magnetic field (50 Hz) does not damage DNA and hence is not mutagenic in this assay and that there was also no influence on the DNA damaging capacity of the used mutagens.

Genotoxicity testing of 3,4,5-trimethylfuran-2(5H)-one, a compound from plant-derived smoke with germination inhibitory activity.

Plant-derived smoke and certain smoke compounds improve seed germination and enhance seedling growth of many species. Thus, smoke-infused water and the active smoke-derived compounds have the potential to be used in different agricultural and horticultural applications. However, despite these interesting and potentially practical properties, it should also be ascertained whether such compounds may pose a health risk, particularly if they are to be used in the production of food or fodder crops. Amongst some of the aspects that would be important to understand are any possible genotoxic properties that the compounds may possess due to potential carry-over effects. Here, we report on a genotoxicity study of 3,4,5-trimethylfuran-2(5H)-one, a compound from plant-derived smoke previously shown to have germination inhibitory activity. Using two in vitro tests, namely the bacterial VITOTOX® test (with/without S9 metabolic activation) and the cytome assay on human C3A cells, no genotoxicity or toxicity was found. Furthermore, these results support a previous study where a related smoke-derived compound with germination promoting properties was investigated.

1,2,3,4,8,9,10,11-octahydrobenzo[j]phenanthridine-7,12-diones as new leads against Mycobacterium tuberculosis.

Tuberculosis (TB) continues to be a worldwide health problem with over 1.4 million deaths each year. Despite efforts to develop more effective vaccines, more reliable diagnostics, and chemotherapeutics, tuberculosis remains a threat to global health, fueled by the HIV pandemic and the rapid generation of drug resistance. The exploration of novel drugs to serve as a companion drug for existing drugs is of paramount importance. As part of our program to design new 2-aza-anthraquinones with antimycobacterial activity, various tetrahydro- and octahydrobenzo[j]phenanthridinediones were synthesized. These compounds showed high in vitro potency against Mycobacterium tuberculosis, the etiological agent of TB and against other clinically relevant mycobacterial species at submicromolar concentrations. The susceptibility of a multidrug resistant strain toward these compounds and their ability to target intracellular replicating Mycobacterium tuberculosis was demonstrated. Next to the acute toxicity, the genotoxicity of these compounds was investigated. Often overlooked in studies, genotoxicity could be dismissed for the investigated compounds, making them a promising scaffold in TB drug research.

Biological evaluation of bisbenzaldehydes against four Mycobacterium species.

A series of bisbenzaldehydes and structurally related analogs, conveniently synthesized via microwave-assisted reactions, were evaluated in vitro against drug susceptible and multi-drug resistant Mycobacterium tuberculosis, against virulent Mycobacterium bovis, against Mycobacterium ulcerans and against two Mycobacterium avium subspecies. Among the 33 substances that were tested, compound 12, i.e. 4,4'-[1,12-dodecanediyl(oxy)]bisbenzaldehyde, emerged as the most promising hit. Its activity was further confirmed in an intracellular growth inhibition assay of M. tb in murine J774 A.1 macrophages. None of the compounds showed significant cytotoxicity on human C3A hepatocytes in a neutral red dye uptake assay and no genotoxicity or mutagenicity was observed as demonstrated by a VITOTOX™ test and confirmed with a comet assay.

Straightforward palladium-mediated synthesis and biological evaluation of benzo[j]phenanthridine-7,12-diones as anti-tuberculosis agents.

In 1991, WHO recognized the resurgence of tuberculosis as a global health problem. Although modern chemotherapy is effective against the causative pathogen Mycobacterium tuberculosis, the current drug regimens have failed to eradicate the disease. The success of the pathogen, partially attributed to drug resistance, necessitates the development of novel anti-tuberculosis drugs. Benzo[j]phenanthridine-7,12-diones, tetracyclic derivatives of the natural product benz[g]isoquinoline-5,10-dione, were conveniently synthesized via palladium-catalyzed intramolecular cyclization of N-methanesulfonyl-3-bromo-2-(arylamino)methyl-1,4-naphthoquinones. Here we report on the bioactivity of eight benzo[j]phenanthridine-7,12-dione derivatives as candidate drug molecules against M. tuberculosis and on their cytotoxicity on C3A human hepatocytes. The strongest antimicrobial activity (as detected by growth inhibition of bacteria, using luminometry and BACTEC 460-TB) and lowest cytotoxicity was found for 3-methylbenzo[j]phenanthridine-7,12-dione 5e, which was also effective in targeting intracellular M. tuberculosis (in murine J774 macrophages) and was not genotoxic for C3A hepatocytes.

Genotoxicity investigation of ELF-magnetic fields in Salmonella typhimurium with the sensitive SOS-based VITOTOX test.

We performed a genotoxicity investigation of extremely low-frequency (ELF) magnetic fields (MFs, 50 Hz, 100 and 500 µT, 1 and 2 h exposure) alone and in combination with known chemical mutagens using the VITOTOX test. This test is a very sensitive reporter assay of Salmonella typhimurium bacteria based on the SOS response. Our study showed that ELF-MFs do not induce SOS-based mutagenicity in S. typhimurium bacteria and do not show any synergetic effect when combined with chemical mutagens.

Eco-, geno- and human toxicology of bio-active nanoparticles for biomedical applications.

Gene delivery has become an increasingly important strategy for treating a variety of human diseases, including infections, genetic disorders and tumours. To avoid the difficulties of using viral carriers, more and more non-viral gene delivery nanoparticles are developed. Among these new approaches polyethylene imine (PEI) is currently considered as one of the most effective polymer based method solution and considered as the gold standard. The toxicity of nanoparticles is a major concern when used for medical application. In this study we chose two nanoparticles for an in depth toxicological and ecotoxicological evaluation, one well characterized, PEI, and another novel polymer, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA). In the present study we have assessed the toxicity of these cation nanoparticles as such and of the polyplexes - nanoparticles covered with DNA. As these nanoparticles are also frequently used in high volumes in various industries and as such may enter in the environment, we also made an initial assessment of ecotoxicological effects assessment. The following nanoparticles related aspects have been studied during the project: development and characterization, ecotoxicity, general toxicity and specific toxicity. To this end a battery of different tests was used. The conclusion of these tests is that toxicity is varying between different nanoparticles and between different DNA covering ratios. In general, in the different systems tested, the PEI polymer is more toxic than the PDMAEMA polymer. The same difference is seen for the polyplexes and the higher the charge ratio, the more toxic are the polyplexes. Our study also clearly shows the need for a broad spectrum of toxicity assays for a comprehensive risk assessment. Our study has performed such a comprehensive analysis of two biomedical nanoparticles.

A database of IC50 values and principal component analysis of results from six basal cytotoxicity assays, for use in the modelling of the in vivo and in vitro data of the EU ACuteTox project.

The main aim of the ACuteTox project (part of the EU 6th Framework programme) is to demonstrate that animal tests for acute systemic toxicity can be replaced by alternative in vitro assays. In this project, data for 97 reference chemicals were collected in the AcuBase database, designed to handle deposited in vitro and in vivo (human and animal) data. To demonstrate the applicability of in vitro basal cytotoxicity tests and in vitro-in vivo modelling, it was deemed necessary to obtain data that were generated via defined standard operating procedures. The molar basal cytotoxicity IC50 values (the 50% inhibitory concentrations for the endpoint measured) for a mouse fibroblast cell line (3T3), a human hepatic cell line (HepG2), a rat hepatic cell line (Fa32), and a human neutrophil cell line (HL-60), were compared, and gave an R(2) correlation of 0.83. To identify chemicals that showed differential cytotoxicity to the various cell types involved, principal component analysis (PCA) was undertaken independently, once all the results had been returned. This showed that colchicine, cycloheximide, digoxin, 5-fluorouracil and hexachlorobenzene gave the lowest correlations with the first score vector of the PCA. The results presented are to be used to identify outliers that need to be further studied via the use of tissue-specific in vitro assays.