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The two-test in vitro battery for genotoxicity testing (Ames and micronucleus) has in the majority of cases replaced the three-test battery (as two-test plus mammalian cell gene mutation assay) for the routine testing of chemicals, pharmaceuticals, cosmetics, and agrochemical metabolites originating from food and feed as well as from water treatment. The guidance for testing agrochemical groundwater metabolites, however, still relies on the three-test battery. Data collated in this study from 18 plant protection and related materials highlights the disparity between the often negative Ames and in vitro chromosome aberration data and frequently positive in vitro mammalian cell gene mutation assays. Sixteen of the 18 collated materials with complete datasets were Ames negative, and overall had negative outcomes in in vitro chromosome damage tests (weight of evidence from multiple tests). Mammalian cell gene mutation assays (HPRT and/or mouse lymphoma assay (MLA)) were positive in at least one test for every material with this data. Where both MLA and HPRT tests were performed on the same material, the HPRT seemed to give fewer positive responses. In vivo follow-up tests included combinations of comet assays, unscheduled DNA synthesis, and transgenic rodent gene mutation assays, all gave negative outcomes. The inclusion of mammalian cell gene mutation assays in a three-test battery for groundwater metabolites is therefore not justified and leads to unnecessary in vivo follow-up testing., (© The Author(s) 2024. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.)

We demonstrate a single-step method for the generation of collagen and poly-l-Lysine (PLL) micropatterns on a poly(ethylene glycol) (PEG) functionalized glass surface for cell based assays. The method involves establishing a reliable silanization method to create an effective non-adhesive PEG layer on glass that inhibits cell attachment, followed by the spotting of collagen or PLL solutions using non-contact piezoelectric printing. We show for the first time that the spotted protein micropatterns remain stable on the PEG surface even after extensive washing, thus significantly simplifying protein pattern formation. We found that adherence and spreading of NIH-3T3 fibroblasts was confined to PLL and collagen areas of the micropatterns. In contrast, primary rat hepatocytes adhered and spread only on collagen micropatterns, where they formed uniform, well defined functionally active cell arrays. The differing affinity of hepatocytes and NIH-3T3 fibroblasts for collagen and PLL patterns was used to develop a simple technique for creating a co-culture of the two cell types. This has the potential to form structured arrays that mimic the in vivo hepatic environment and is easily integrated within a miniaturized analytical platform for developing high throughput toxicity analysis in vitro., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The application of toxicogenomics as a predictive tool for chemical risk assessment has been under evaluation by the toxicology community for more than a decade. However, it predominately remains a tool for investigative research rather than for regulatory risk assessment. In this study, we assessed whether the current generation of microarray technology in combination with an in vitro experimental design was capable of generating robust, reproducible data of sufficient quality to show promise as a tool for regulatory risk assessment. To this end, we designed a prospective collaborative study to determine the level of inter- and intra-laboratory reproducibility between three independent laboratories. All test centres (TCs) adopted the same protocols for all aspects of the toxicogenomic experiment including cell culture, chemical exposure, RNA extraction, microarray data generation and analysis. As a case study, the genotoxic carcinogen benzo[a]pyrene (B[a]P) and the human hepatoma cell line HepG2 were used to generate three comparable toxicogenomic data sets. High levels of technical reproducibility were demonstrated using a widely employed gene expression microarray platform. While differences at the global transcriptome level were observed between the TCs, a common subset of B[a]P responsive genes (n=400 gene probes) was identified at all TCs which included many genes previously reported in the literature as B[a]P responsive. These data show promise that the current generation of microarray technology, in combination with a standard in vitro experimental design, can produce robust data that can be generated reproducibly in independent laboratories. Future work will need to determine whether such reproducible in vitro model(s) can be predictive for a range of toxic chemicals with different mechanisms of action and thus be considered as part of future testing regimes for regulatory risk assessment., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Liver cell lines and primary hepatocytes are becoming increasingly valuable for in vitro toxicogenomic studies, with RT-qPCR enabling the analysis of gene expression profiles following exposure to potential hepatotoxicants. Supporting the accurate normalisation of RT-qPCR data requires the identification of reference genes which have stable expression during in vitro toxicology studies. Therefore, we performed a comprehensive analysis of reference gene stability in two routinely used cell types, (HepG2 cells and primary rat hepatocytes), and two in vitro culture systems, (2D monolayer and 3D scaffolds). A robust reference gene validation strategy was performed, consisting of geNorm analysis, to test for pair wise variation in gene expression, and statistical analysis using analysis of variance. This strategy identified stable reference genes with respect to acetaminophen treatment and time in HepG2 cells (GAPDH and PPIA), and with respect to acetaminophen treatment and culture condition in primary hepatocytes (18S rRNA and α-tubulin). Following the selection of reference genes, the novel target genes E2F7 and IL-11RA were identified as potential toxicity biomarkers for acetaminophen treatment. We conclude that accurate quantification of gene expression requires the use of a validated normalisation strategy for each species and experimental system employed., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

This study investigates whether the previous observation that quercetin increases the transport of PhIP through Caco-2 monolayers in vitro could be confirmed in an in vivo rat model. Co-administration of 1.45 micromol PhIP/kg bw and 30 micromol quercetin/kg bw significantly increased the blood AUC(0-8h) of PhIP in rats to 131+/-14% of the AUC(0-8h) for rats dosed with PhIP alone. Significantly increased blood PhIP levels were detected at 15, 30, 45 and 180 min. At 4 and 8h post-dosing a difference in the PhIP levels in the blood between the two treatment groups was no longer observed. In vitro and in silico modeling of PhIP transport using Caco-2 cells and a previously described kinetic model for PhIP transport revealed that the relative increase in PhIP transport caused by quercetin is dependent on the concentration of the two compounds. When substituting the PhIP and quercetin concentrations used in the in vivo experiment in the kinetic model, an effect of quercetin on PhIP transport was predicted that matches the actual effect of 131% observed in vivo. It is concluded that quercetin increases the bioavailability of the pro-carcinogen PhIP in rats pointing at a potential adverse effect of this supposed beneficial food ingredient.

This study describes and kinetically models the effect of flavonoid mixtures on PhIP transport through Caco-2 monolayers. Previously it was shown that quercetin, luteolin, naringenin and myricetin increase the apical to basolateral PhIP transport in Caco-2 monolayers. In this study, apigenin was shown to exert a similar effect with an apparent K(i) value of 10.8 microM. Additional experiments revealed that several binary flavonoid mixtures and one mixture containing all five model flavonoids increased the apical to basolateral PhIP transport through the Caco-2 monolayer. Assuming competitive inhibition of the apparent active transporter by the flavonoids and concentration-additivity for their inhibiting effect, the kinetic model previously developed to describe the effect of the individual flavonoids on PhIP transport, could be extended and adequately describes the experimental values obtained for the flavonoid mixtures. We conclude that combinations of flavonoids increase the transport of PhIP and do so by interacting in an additive way with the active transport of PhIP. This flavonoid-mediated increase in PhIP transport through Caco-2 monolayers may point at a possible increased bioavailability of PhIP in the presence of flavonoid mixtures in the in vivo situation. This would imply an adverse effect of these supposed beneficial food ingredients.

The present study describes the effect of different flavonoids on the absorption of the pro-carcinogen PhIP through Caco-2 monolayers and the development of an in silico model describing this process taking into account passive diffusion and active transport of PhIP. Various flavonoids stimulated the apical to basolateral PhIP transport. Using the in silico model for flavone, kaempferol and chrysoeriol, the apparent Ki value for inhibition of the active transport to the apical side was estimated to be below 53 muM and for morin, robinetin and taxifolin between 164 and 268 microM. For myricetin, luteolin, naringenin and quercetin, the apparent Ki values were determined more accurately and amounted to 37.3, 12.2, 11.7 and 5.6 microM respectively. Additional experiments revealed that the apical to basolateral PhIP transport was also increased in the presence of a typical BCRP or MRP inhibitor with apparent Ki values in the same range as those of the flavonoids. This observation together with the fact that flavonoids are known to be inhibitors of MRPs and BCRP, corroborates that inhibition of these apical membrane transporters is involved in the flavonoid-mediated increased apical to basolateral PhIP transport. Based on the apparent Ki values obtained, it is concluded that the flavonols, at the levels present in the regular Western diet, are capable of stimulating the transport of PhIP through Caco-2 monolayers from the apical to the basolateral compartment. This points to flavonoid-mediated stimulation of the bioavailability of PhIP and, thus, a possible adverse effect of these supposed beneficial food ingredients.

The transcellular transport of ingested food ingredients across the intestinal epithelial barrier is an important factor determining bioavailability upon oral intake. This transcellular transport of many chemicals, food ingredients, drugs or toxic compounds over the intestinal epithelium can be highly dependent on the activity of membrane bound ATP binding cassette (ABC) transport proteins, able to export the compounds from the intestinal cells. The present review describes the ABC transporters involved in the efflux of bioactive compounds from the intestinal cells, either to the basolateral blood side, facilitating absorption, or back into the intestinal lumen, reducing bioavailability. The role of the ABC transporters in intestinal transcellular uptake also implies a role for inhibitors of these transporters in modulation of the bioavailability upon oral uptake. The present paper focuses on the role of flavonoids as important modulators or substrates of intestinal ABC transport proteins. Several examples of such an effect of flavonoids are presented. It can be concluded that flavonoid-mediated inhibition of ABC transporters may affect the bioavailability of drugs, bioactive food ingredients and/or food-borne toxic compounds upon oral uptake. All together it appears that the flavonoid-mediated interactions at the level of the intestinal ABC transport proteins may be an important mechanism for unexpected food-drug, food-toxin or food-food interactions. The overview also indicates that future studies should focus on i) in vivo validation of the flavonoid-mediated effects on bioavailability of drugs, toxins and beneficial bioactive food ingredients detected in in vitro models, and on ii) the role of flavonoid phase II metabolism in modulating the activity of the flavonoids to act as ABC transporter inhibitors and/or substrates.

The effect of the flavonoid myricetin on the transport of the pro-carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through differentiated Caco-2 monolayers, a model for the intestinal epithelium, is described. Myricetin causes an increase of the transport of PhIP from the apical to the basolateral compartment. This effect was observed at physiologically relevant concentrations of PhIP and myricetin. Cyclosporin A (MRP2 inhibitor) but not PSC833 (P-gp inhibitor) showed a similar effect on PhIP transport. The results indicate that myricetin induces an increased basolateral uptake of the pro-carcinogen PhIP, in part through inhibition of the MRP2 mediated excretion of PhIP from the intestinal cells back to the lumen.

The present review focuses on the mechanisms of mutagenic action and the carcinogenic risk of two categories of botanical ingredients, namely the flavonoids with quercetin as an important bioactive representative, and the alkenylbenzenes, namely safrole, methyleugenol and estragole. For quercetin a metabolic pathway for activation to DNA-reactive species may include enzymatic and/or chemical oxidation of quercetin to quercetin ortho-quinone, followed by isomerisation of the ortho-quinone to quinone methides. These quinone methides are suggested to be the active alkylating DNA-reactive intermediates. Recent results have demonstrated the formation of quercetin DNA adducts in exposed cells in vitro. The question that remains to be answered is why these genotoxic characteristics of quercetin are not reflected by carcinogenicity. This might in part be related to the transient nature of quercetin quinone methide adducts, and suggests that stability and/or repair of DNA adducts may need increased attention in in vitro genotoxicity studies. Thus, in vitro mutagenicity studies should put more emphasis on the transient nature of the DNA adducts responsible for the mutagenicity in vitro, since this transient nature of the formed DNA adducts may play an essential role in whether the genotoxicity observed in vitro will have any impact in vivo. For alkenylbenzenes the ultimate electrophilic and carcinogenic metabolites are the carbocations formed upon degradation of their 1'-sulfooxy derivatives, so bioactivation of the alkenylbenzenes to their ultimate carcinogens requires the involvement of cytochromes P450 and sulfotransferases. Identification of the cytochrome P450 isoenzymes involved in bioactivation of the alkenylbenzenes identifies the groups within the population possibly at increased risk, due to life style factors or genetic polymorphisms leading to rapid metaboliser phenotypes. Furthermore, toxicokinetics for conversion of the alkenylbenzenes to their carcinogenic metabolites and kinetics for repair of the DNA adducts formed provide other important aspects that have to be taken into account in the high to low dose risk extrapolation in the risk assessment on alkenylbenzenes. Altogether the present review stresses that species differences and mechanistic data have to be taken into account and that new mechanism- and toxicokinetic-based methods and models are required for cancer risk extrapolation from high dose experimental animal data to low dose carcinogenic risks for man.

Previous studies have indicated that a sizable fraction of adult human peripheral blood B cells may express IgM receptors encoded by somatically mutated V regions. From these studies it was uniquely associated with the peripheral blood B cell compartment, was uniquely associated with the peripheral blood B cell compartment, associated with particular VH gene segments and/or B cell subpopulations. We have addressed these issues by analyzing > 80 VH5 and VH6-encoded mu transcripts from unseparated peripheral blood, tonsil, and spleen B cells, as well as from B cells separated on the basis of CD5 Ag expression. The results demonstrate that somatically mutated VH5 and VH6 regions are ubiquitously expressed in IgM-bearing B cells in all peripheral adult human lymphoid organs, and that the occurrence of somatic mutations does not segregate with either CD5+ or CD5- B cell populations. The distribution and nature of mutations, as well as the occurrence of clonally related but divergent transcripts suggests that at least some of the mutations were selected by Ag.

The Ig VH regions of virtually all human pathogenic cold agglutinin (CA) anti-i/l autoantibodies are encoded by a single Ig VH gene segment, VH4.21, in conjunction with a highly variable CDR3 structure. The anti-I specificity is often associated with V kappa III-encoded L chains, whereas anti-i autoantibodies appear to use a broader array of kappa and lambda VL gene segments. B cells expressing VH4.21 are abundantly present in adult lymphoid tissues from healthy individuals but their relationship with B cells secreting pathogenic CA is unknown. Herein we have analyzed the distribution of VH4.21-expressing B cells in fetal, neonatal, and adult B cell populations using the monoclonal anti-VH4.21 antibody 9G4. In addition, we have analyzed the anti-i and anti-I binding capacity and V regions of 19 VH4.21-encoded mAb secreted by cord and adult blood-derived cell lines from healthy individuals. The results show that VH4.21 expressing B cells are overrepresented in all repertoires studied and are evenly distributed over cord blood CD5+ and CD5- B cell populations. VH4.21-encoded H chains strongly predispose for anti-i binding capacity, regardless of the VL regions or H chain CDR3 structure. The avidity of some of these antibodies was similar to those of pathogenic CA. In addition, we found evidence for monospecific anti-I binding in antibodies encoded by other members of the VH4 gene family. We conclude that VH4.21-encoded antibodies with anti-i specificity from the normal B cell repertoire mirror their pathogenic counterparts and that naturally occurring anti-I antibodies may be encoded by a more diverse array of VH4 genes.

We have fractionated human tonsillar B cells on the basis of CD5 expression and determined the nucleotide sequences of immunoglobin light chain variable (V) regions encoded by the single member of the V chi 4 gene family in both CD5+ and CD5- populations. The majority of cDNA from both CD5+ and CD5- B cells populations harbored somatic mutations. Thus, human tonsillar CD5+ B cells, unlike their murine counterparts, are capable of activating their somatic hypermutation mechanism, resulting in the accumulation of somatic mutation in the VL regions.

VH gene segments expressed in a panel of monoclonal human CD5 B cell lines have been positioned on the IgH locus by deletion mapping. The analysis yielded a relative order of VH fragments of the VH2, VH4, VH5, and VH6 gene families that was consistent with, and provided a further refinement of existing maps of the human IgH locus. We demonstrate that four of six VH gene segments expressed in the CD5 B cell lines map > 500 kb from the cluster of JH segments. Two of the gene segments, positioned at approximately 850 kb (58p2) and approximately 500 kb (1-9III) from the JH segments, respectively, belong to the previously identified small cohort of second trimester fetal VH gene segments. The data show that JH proximity is not the sole determinant of restricted VH gene utilization in early human ontogeny.

We have shown that the restricted repertoire of VH genes expressed in second trimester human fetal liver is not solely determined by JH proximity. Furthermore, by following the fate of two VH gene segments in different B-cell repertoires, we have provided evidence that multiple factors contribute to the frequency with which individual VH genes are utilized. We found that the repertoire of adult blood IgM-bearing B cells contains a high proportion of B lymphocytes that express extensively mutated VH genes. Finally, we show that somatically-mutated variants of particular VH and VL genes that, in germline configuration, are frequently found in the early B-cell repertoire and in natural autoantibodies, encode pathogenic IgG autoantibodies characteristic of human SLE. These VH and VL genes harbor all the characteristics of an antigen-driven B-cell activation and selection process.

Previous studies on the repertoire of Ig VH genes utilized in the malignant cells of chronic lymphocytic leukemia (CLL) have suggested a non-random expression pattern. In particular, individual genes from the VH1, VH5, and VH6 gene families have been frequently found in CLL. With regard to other VH gene families, including the large VH3 family which is expressed in greater than 50% of CLL cases, it is unknown whether CLL cells utilize either a broad or a restricted repertoire of VH genes. In the present paper, we analyzed the VH genes expressed in a collection of 11 CLL cases. The results of these experiments demonstrate that there is no apparent restriction in the usage of individual members of the VH3 gene families in CLL.

Human B lymphocytes that bear the CD5 antigen are relatively abundant in early ontogeny and comprise a small fraction of the B cell population in adults. The CD5 B cell subset has attracted much attention because of its possible involvement in autoimmune disease and certain B cell malignancies. To begin to understand the role of CD5 B cells in disease processes, we have generated a panel of ten human monoclonal B cell lines selected for expression of the CD5 antigen. These cell lines were obtained by Epstein-Barr virus transformation of B lymphocytes isolated from the spleen, liver and bone marrow of a 19-week-old fetus, from cord blood and from peripheral blood of healthy volunteers. In addition, one cell line was isolated from the spleen of a patient with chronic lymphocytic leukemia. Here, we describe the antibody and immunoglobulin VH gene repertoire of this panel of CD5 B cell lines. The results of these experiments show that (a) some but not all CD5 B cell lines secrete polyreactive antibodies that bind to a variety of self- and xenoantigens and (b) members of the small VH4, VH5 and VH6 gene families are overrepresented in this panel of cell lines. Nucleotide sequence analysis revealed the expression of VH gene elements that have been previously reported in the preimmune B cell repertoire, in CD5 B cell tumors and in polyreactive antibodies.

We have studied frequencies of VH gene utilization in a panel of monoclonal Epstein-Barr virus (EBV)-transformed B cell lines derived from human adult and fetal tissues as well as in monoclonal B cells obtained from fresh chronic lymphocytic leukemia (CLL) samples. The results show that IgM-secreting EBV cell lines from both fetal and adult tissues utilize VH genes from particular families roughly in proportion to estimated family size, suggesting that the repertoire of sigM-positive B cells in both fetal and adult organs is 'normalized' with respect to the V(H) gene family. In contrast, we find a highly biased pattern of VH gene expression in CLLs. The significance of these findings is discussed in the context of mechanisms that could be involved in normal B cell repertoire development and in the process of malignant transformation of precursors of CLL.