A high-resolution screening platform, coupling online affinity detection for mammalian cytochrome P450s (Cyt P450s) to gradient reversed-phase high-performance liquid chromatography (HPLC), is described. To this end, the onlineCyt P450 enzyme affinity detection (EAD) system was optimized for enzyme (β-NF-induced rat liver microsomes), probe substrate (ethoxyresorufine), and organic modifier (methanol or acetonitrile). The optimized Cyt P450 EAD system has first been evaluated in a flow injection analysis (FIA) mode with 7 known ligands of Cyt P450 1A1/1A2 (β-naphthoflavone, ßnaphthoflavone, ellipticine, 9-hydroxy-ellipticine, fluvoxamine, caffein, and phenacetin). Subsequently, IC 50 valueswere online in FIA-mode determined and compared with those obtained with standardmicrosomal assay conditions. The IC 50 values obtained with the online Cyt P450 EAD system agreed well with the IC 50 values obtained in the standard assays. For highaffinity ligands ofCyt P450 1A1/1A2, detection limits of 1 to 3 pmol injected (n= 3; signal to noise [S/N] = 3) were obtained. The individual inhibitory properties of ligands in mixtures of the ligands were subsequently investigated using an optimized Cyt P450 EAD system online coupled to gradient HPLC. Using the integrated online gradient HPLC Cyt P450 EAD platform, detection limits of 10 to 25 pmol injected (n= 1; S/N= 3) were obtained for high-affinity ligands. It is concluded that this novel screening technology offers new perspectives for rapid and sensitive screening of individual compounds in mixtures exhibiting affinity for liver microsomal Cyt P450s.

Exposure of humans and wildlife to xenobiotics, such as halogenated biphenyls, that interfere with the endogenous estrogen balance may lead to endocrine disruption. Such compounds may either mimic or block estradiol's action by agonistic or antagonistic action, respectively. They may also affect endogenous estradiol concentrations by induction or inhibition of enzymes that metabolize estradiol. In the present study, we demonstrate that estrogenic metabolites of two brominated biphenyls, 2,2'-dibromobiphenyl (2,2'-DBB) and 4,4'-dibromobiphenyl (4,4'-DBB), are formed by rat liver microsomal cytochrome P450 (CYP) activity. Bioactivation of 2,2'-DBB and 4,4'-DBB yielded various mono- and dihydroxylated bromobiphenyl metabolites, which were collected by preparative HPLC and analyzed by LC/MS. Several of the metabolites bound to the estrogen receptor (ER) activated the ER and inhibited human estrogen sulfotransferase (hEST). Seven monohydroxylated metabolites were positively identified using synthetic monohydroxylated reference compounds. These synthetic monohydroxylated bromobiphenyls also bound to and activated the ER and inhibited hEST. The highest ER affinity was observed for 4-OH-2,2'-DBB, with an EC50 of 6.6 nM. The highest ER activation was observed for 4-OH-3,4'-DBB (EC50 of 74 nM) while 4-OH-4'-MBB and 4-OH-2,2'-DBB induced a supramaximal (as compared to estradiol) ER activation. The strongest hEST inhibition was found with 4-OH-3,4'-DBB (EC50 = 40 nM). In conclusion, we show that two dibrominated biphenyls are bioactivated by CYP activity into very potent estrogenic metabolites and inhibitors of hEST. These findings are of vital importance for accurate risk assessment of exposure to environmental contaminants, such as halogenated biphenyls. Neglecting bioactivation through biotransformation will lead to underestimation of health risks of this class of xenobiotics.

Coumestrol is a well-known ligand for the estrogen receptor (ER). The compound itself is fluorescent, and its fluorescence intensity at 408 nm increases upon binding to the ER. Here we describe a novel binding assay in 96-well plate format for estrogenic compounds, based on the competition between fluorescent coumestrol, and estrogenic compounds for binding to the ligand binding domain (LBD) of the ER-alpha. Displacement of coumestrol was measured as a decrease in fluorescence intensity using a Victor-1420 multilabel reader. Competitive binding curves for the well-known estrogenic Compounds, 17 beta-estradiol (E-2), ethinylestradiol, 4-nonylphenol, 4-octylphenol, genistein, bisphenol A, tamoxifen and diethyl-stilbestrol were constructed by using 7-10 different concentrations of the compounds and a fixed concentration of ER-alpha-LBD (14 nmol) and coumestrol (100 nmol). IC50) values and relative potencies (compared to E-2) of the estrogenic compounds were determined. The assay was validated by comparing the relative potencies to those from standard radioligand binding assays in the literature. Within day and between day variations were determined and the performance of the assay was assessed by determining the coefficients of variation and Z ' values. The present fluorescent binding assay has proven to be fast and easy, and allows accurately quantifying the binding affinity of estrogenic ligands. The method is also suitable as a high-throughput screening assay for ER ligands.

A novel approach is presented for studying the chemical interaction between receptor binding sites and ligands. Monohydroxylated polyaromatic compounds were found to be environmentally sensitive ligands when applying a special mode of fluorescence: fluorescence line-narrowing spectroscopy (FLNS). With this technique, solvent dependencies and ligand-receptor interactions can be studied in great detail, due to the high spectral resolution and the fact that at cryogenic temperatures (4 K), no solvent reorientation effects complicate the interpretation. The FLN spectrum of a ligand bound to the receptor is compared to the spectra of the free ligand in solvent mixtures that mimic the functionalities present within the receptor's binding site. It is shown that for the well-known estrogen receptor (ER), the orientations of two xenoestrogenic ligands 3- and 9-hydroxybenzo[a]pyrene (3- and 9-OH-BaP) can be determined. The FLN results clearly indicate that an H-bond accepted by HIS524 plays a major role in the binding of these ligands to the ER. Furthermore, the spectra indicated a pi-pi stacking aromatic interaction for 9-OH-BaP with PHE404. These results are in line with molecular modeling studies published earlier.

The objective of the present study was to evaluate a comprehensive set of urinary biomarkers for oxidative damage to lipids, proteins and DNA, in man. Eighteen moderately trained males (mean age 24.6±0.7) exercised 60 min at 70% of maximal O2 uptake on a cycle ergometer. Urine fractions for 12 h were collected 1 day before, and for 3 consecutive days after exercise. As biomarkers of lipid peroxidation, 8 aldehydes (i.e. propanal, butanal, pentanal, hexanal, heptanal, octanal, nonanal and malondialdehyde—MDA)and acetone were analyzed in urines by gas chromatography with electron capture detection (GC-ECD). As a biomarker of protein oxidation, o,o′-dityrosine was analyzed in urine samples by a recently developed isotope dilution HPLC-atmospheric pressure chemical ionization (APCI)-tandem-mass spectrometry (HPLC-APCI-MS/MS) methodology. As a biomarker of oxidative DNA damage, urinary excretion of 8-hydroxy-2′-deoxyguanosine (8-OHdG) was measured by an ELISA method. On the day of exercise, significant increases were observed in urinary excretions of acetone ( p<0.025, n=18) and butanal ( p<0.01, n=18) in the 12 h daytime fractions compared to the daytime fraction before exercise. The urinary acetone excretion was also significantly ( p<0.05) increased on the 1st day after exercise. Octanal and nonanal were increased in the daytime urine fraction on the 2nd day after exercise. However, these increases were of borderline significance ( p=0.09 and p=0.07, respectively). Significantly elevated urinary o,o′-dityrosine amounts were observed in the daytime fraction on the day of exercise ( p<0.025) and on the 1st day after exercise ( p=0.07) compared to the before exercise daytime fraction. Excretion of urinary 8-OHdG was statistically significantly increased in the daytime fractions on the day of exercise ( p=0.07) and on the 1st day after exercise ( p<0.025) compared to before exercise daytime fraction. Increases in urinary excretions of acetone, propanal, pentanal, MDA and 8-OHdG significantly correlated with training status (hours of exercise/week) of the volunteers, while o,o′-dityrosine did not. To our knowledge, the present study is the first to evaluate a multi-parameter non-invasive biomarker set for damage to three main cellular targets of ROS. It shows that 1 h of exercise may already induce oxidative damage in moderately trained individuals and that the chosen urinary biomarkers are sensitive enough to monitor such damage.

Exposure to environmental estrogens has been proposed as a risk factor for disruption of reproductive development and tumorigenesis of humans and wildlife (McLachlan, J. A.; Korach, K. S.; Newbold, R. R.; Degen, G. H. Diethylstilbestrol and other estrogens in the environment. Fundam. Appl. Toxicol. 1984, 4, 686-691). In recent years, many structurally diverse environmental compounds have been identified as estrogens. A reliable computational method for determining estrogen receptor (ER) binding affinity is of great value for the prediction of estrogenic activity of such compounds and their metabolites. In the presented study, a computational model was developed for prediction of binding affinities of ligands to the ERalpha isoform, using MD simulations in combination with the linear interaction energy (LIE) approach. The linear interaction energy approximation was first described by Aqvist et al. (Aqvist, J.; Medina, C.; Samuelsson, J. E. A new method for predicting binding affinity in computer-aided drug design. Protein Eng. 1994, 7, 385-391) and relies on the assumption that the binding free energy (DeltaG) depends linearly on changes in the van der Waals and electrostatic energy of the system. In the present study, MD simulations of ligands in the ERalpha ligand binding domain (LBD) (Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.; Kushner, P. J.; Agard, D. A.; Greene, G. L. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 1998, 95, 927-937), as well as ligands free in water, were carried out using the Amber 6.0 force field (http://amber.scripps.edu/). Contrary to previous LIE methods, we took into account every possible orientation of the ligands in the LBD and weighted the contribution of each orientation to the total binding affinity according to a Boltzman distribution. The training set (n = 19) contained estradiol (E2), the synthetic estrogens diethylstilbestrol (DES) and 11beta-chloroethylestradiol (E2-Cl), 16alpha-hydroxy-E2 (estriol, EST), the phytoestrogens genistein (GEN), 8-prenylnaringenin (8PN), and zearalenon (ZEA), four derivatives of benz[a]antracene-3,9-diol, and eight estrogenic monohydroxylated PAH metabolites. We obtained an excellent linear correlation (r(2) = 0.94) between experimental (competitive ER binding assay) and calculated binding energies, with K(d) values ranging from 0.15 mM to 30 pM, a 5 000 000-fold difference in binding affinity. Subsequently, a test set (n = 12) was used to examine the predictive value of our model. This set consisted of the synthetic estrogen 5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol (THC), daidzein (DAI), equol (EQU) and apigenin (API), chlordecone (KEP), progesterone (PRG), several mono- and dihydroxylated PAH metabolites, and two brominated biphenyls. The predicted binding affinities of these estrogenic compounds were in very good agreement with the experimental values (average deviation of 0.61 +/- 0.4 kcal/mol). In conclusion, our LIE model provides a very good method for prediction of absolute ligand binding affinities, as well as binding orientation of ligands.

A study was performed to optimize sample preparation and application of three in vitro assays for measuring estrogenic potency in environmental extracts. The three assays applied were an estrogen receptor (ER)-binding assay and two reporter gene effect assays: a yeast estrogen screen (YES) and the ER-mediated chemically activated luciferase gene expression (ER-CALUX) assay. All assays were able to detect estrogenicity, but the amounts of material needed for the assays differed greatly between the three assays (ER-binding assay >> YES > ER-CALUX). In addition, in the ER-binding assay, both agonists and antagonists give an estrogenic response, resulting in higher estradiol equivalency (EEQ) levels than both the ER-CALUX and the YES assay for the same samples. The EEQs found in wastewater treatment plants (WTPs) with the ER-CALUX assay were in the range of 4 to 440 and 0.11 to 59 pmol/L for influent and effluent, respectively. Water extracts from four large rivers had levels ranging from 0.25 to 1.72 pmol/L. Extracts from suspended matter and sludge contained estrogenic potency of 0.26 to 2.49 and 1.6 to 41 pmol EEQ/g dry weight, respectively. In WTPs, the average reduction of estrogenic potency in effluent compared to influent was 90 to 95% in municipal WTPs and about 50% in industrial WTPs. In influent, 30% of the ER-CALUX activity could not be explained by the calculated potencies based on chemical analysis of a number of known (xeno)estrogens; in effluent the unexplained fraction was 80%. These first results of analyzing estrogenic potency in WTP water and surface water in The Netherlands indicate that further studies are warranted to investigate the actual risks for aquatic systems.

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionORIGINAL ARTICLEThis notice is a correctionSimulations of the Estrogen Receptor Ligand-Binding Domain: Affinity of Natural Ligands and Xenoestrogens.B. Chris Oostenbrink, Jed W. Pitera, Marola M. H. van Lipzig, John H. N. Meerman, and Wilfred F. van GunsterenCite this: J. Med. Chem. 2001, 44, 7, 1124Publication Date (Web):March 7, 2001Publication History Published online7 March 2001Published inissue 1 March 2001https://pubs.acs.org/doi/10.1021/jm010823ehttps://doi.org/10.1021/jm010823ecorrectionACS PublicationsCopyright © 2001 American Chemical Society. This publication is available under these Terms of Use. Request reuse permissions This publication is free to access through this site. Learn MoreArticle Views293Altmetric-Citations1LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail PDF (13 KB) Get e-Alertsclose Get e-Alerts

In the absence of (bio)chemical information concerning the active sites of biotransformation enzymes, both indirect and direct computer modeling techniques can be used to get more insight into the active sites and the mechanisms of action of these enzymes. Indirect modeling or small molecule modeling uses a variety of substrates, inhibitors or metabolic products in order to derive a mold of the active site, while direct modeling or homology modeling uses crystal structures of similar proteins to derive a more or less complete model of the protein including the active site. Knowledge about the shape and properties of the active sites may indicate amino acids important for binding of substrates and/or the activation of substrates or intermediates. In this paper, the requirements and the assumptions for the generation of small molecule models and homology models, and the drawbacks and limitations of these models are discussed. Also some methods for the experimental validation of small molecule models and protein models are mentioned. Examples concerning one of the most important biotransformation enzyme families, viz. cytochrome P450, is indicated and discussed.

We recently reported on the design, synthesis and characterisation of a novel and selective substrate of human cytochrome P450 2D6 (CYP2D6), 7-methoxy-4-(aminomethyl)-coumarin (MAMC). Here, we describe a high-throughput microplate reader assay, which makes use of MAMC as a fluorescent probe for determining the inhibition and activity of CYP2D6 in heterologously expressed systems and human liver microsomes. The high-throughput screening (HTS) assay can be used both in an end-point and real-time configuration, and is easy to use, rapid and sensitive. In addition, end-point measurements by means of flow injection analysis have also successfully been performed. The HTS-assay was validated by performing inhibition experiments for several low- and high-affinity ligands (n=6) of CYP2D6, and comparing the findings to those obtained with the standard O-demethylation assay of dextromethorphan. The results indicate that all compounds tested display competitive inhibition in both the MAMC and dextromethorphan assay, and the K(i) values reveal a very good correlation (R(2)=0.984) between the two assays. To further demonstrate the usefulness of the HTS-assay, IC(50) values of a series of five N-substituted analogs of 3, 4-methylenedioxyamphetamine for CYP2D6 have been determined. The results obtained demonstrate that the current HTS-assay represents a significant improvement over previous assays, with a higher turnover of MAMC and a higher selectivity for CYP2D6.

We have carried out molecular dynamics (MD) simulations and free energy calculations on the alpha-subtype of the human estrogen receptor ligand-binding domain (ERalpha LBD) complexed with a number of known agonists and putative xenoestrogens. Our dynamical simulations of ligand-receptor complexes underscore the highly structured nature of the complex and offer some interesting insights into the structure-activity relationship (SAR) for these ligands. With traditional thermodynamic integration (TI) calculations, we calculate relative binding free energies for three known agonists, in good agreement with experimental values. The sheer number of possible xenoestrogenic compounds makes an approach using traditional free energy calculations unfeasible. Instead, we have made use of a single-step perturbation methodology that allows the calculation of relative free energies for a large number of related polyaromatic hydrocarbons (PAHs) from a single simulation. Our results show good (maximum deviation 3.3 kJ mol(-1)) agreement with experimental data, suggesting the possibility of large-scale xenoestrogen screening in silico to obtain strongly estrogenic compounds for subsequent experimental testing.

A series of six structural analogs of 7-methoxy-4-(aminomethyl)-coumarin (MAMC), a recently developed high-throughput substrate of P450 2D6 (CYP2D6), was synthesized to investigate the influence of N-substitution on the metabolism by cytochrome P450s, as well as on P450 selectivity. The analogs were obtained by introducing alkyl substituents at the amino group of MAMC and by replacing this moiety with a pyridine group. Competition experiments using heterologously expressed CYP2D6 demonstrated that the introduction and elongation of alkyl substituents strongly decreased the IC50 values toward dextromethorphan O-demethylation. Metabolism studies showed that the regioselectivity of metabolism was unaffected by the varying N substituents, as only O-dealkylation of the analogs and no N-dealkylation was observed. In excellent agreement with the competition experiments, metabolism studies also showed that elongation of the alkyl chain dramatically increased the affinity of the compounds toward CYP2D6, as indicated by an up to 100-fold decrease in K-m values. The V-max values displayed a much less pronounced decrease with an increasing N-alkyl chain, resulting in as much as a 30-fold increase in the V-max /K-m value. Interestingly, due to the higher fluorescent yield of the N-alkyl metabolites compared with the metabolite of MAMC, O-dealkylation of N-methyl MAMC by CYP2D6 can be measured with a more than 3-fold higher sensitivity. Studies on P450 selectivity showed that only CYP1A2 and CYP2D6 contribute to the O-dealkylation of the N-alkyl analogs in both heterologously expressed P450s and human liver microsomes. In sharp contrast to CYP2D6, N-alkylation of MAMC did not significantly affect the K-m values of O-dealkylation by CYP1A2, but it did result in higher V-max values. Finally, CYP1A2 also N-dealkylated the analogs.

Previous studies have indicated that isolated preneoplastic rat hepatocytes in vitro fail to induce nuclear p53 protein and fail to block replication in response to genotoxic compounds. This suggests that defects in the protection of genomic integrity are part of their premalignant character. In the present study, we have investigated if similar defects occur in vivo. Preneoplastic glutathione-S-transferase (GST) 7-7-positive foci were induced in male Wistar rats by diethylnitrosamine (DEN) initiation and promotion with 2-acetylaminofluorene (2-AAF)/partial hepatectomy (PH). The response to genotoxic damage was studied by X-irradiation. p53 protein was moderately expressed in nuclei in surrounding hepatocytes. This nuclear p53 staining had decreased 2 weeks after 2-AAF treatment. In foci, the protein was detected in the cytoplasm whereas the nuclei were negative. Levels of p21waf1/cip1 protein were high in nuclei and cytoplasm of surrounding hepatocytes, whereas the expression in foci was low. A low level of Mdm2 in nuclei was observed in surrounding liver, while both Mdm2 and Bcl-2 protein were strongly expressed in the cytoplasm in foci. X-ray exposure further induced nuclear expression of p53, p21waf1/cip1, and Mdm2 in surrounding hepatocytes, but focal nuclei were still negative. DNA replication was strongly reduced by X-irradiation in surrounding hepatocytes, but only partially reduced in the foci. These results indicate that the p53 pathway of response to genomic stress is impaired in preneoplastic cells in vivo. This may support their clonal expansion and their further malignant transformation because protection against genetic damage is diminished.

Clonal expansion of initiated cells is an important process in carcinogenesis. Loss of functional p53 protein in initiated, preneoplastic cells might be involved in this process because such a loss would favour cell growth at the expense of normal cells upon exposure to genotoxic compounds. We have tested the hypothesis that p53 is not expressed in preneoplastic cells in the rat liver. Hepatocytes were isolated from livers of 10-week-old female rats that contained foci of preneoplastic hepatocytes, generated by 6-7 weekly injections of diethylnitrosamine (0.15 mmol/kg body wt intraperitoneally (i.p.)), starting 24 h after birth. The mixture of phenotypically normal and preneoplastic hepatocytes was exposed to X-rays or N-acetoxy-acetylaminofluorene (NAAAF), both causing DNA damage directly. At 24 and 48 h after exposure the cells were fixed and double stained for glutathione-S-transferase 7-7 (GST7-7), to identify preneoplastic cells, and p53. The percentage of p53-positive cells was much lower in GST7-7 positive (GST7-7+) than in GST7-7 negative (GST7-7-) hepatocytes. Exposure of cells to X-rays or NAAAF induced p53 in GST7-7- cells after 24 h, but GST7-7+ hepatocytes failed to do so. These results suggest that preneoplastic cells do not express p53 or have an attenuated p53 response to genotoxic treatments. This was confirmed when the cells were exposed to a proteasome inhibitor, PSI, which inhibits p53 degradation: a 12-fold increase in p53-positive cells was found after 48 h in GST7-7- hepatocytes, but in GST7-7+ hepatocytes no increase was observed. The percentage of GST7-7+ hepatocytes among surviving cells was increased after exposure to NAAAF, suggesting that these are more resistant to NAAAF than GST7-7- cells. This was not observed with PSI. These results indicate that preneoplastic hepatocytes have a lower p53 protein content and are not able to increase p53 upon inhibition of p53 breakdown or upon induction of DNA damage. Therefore, loss of p53 may favour clonal expansion of preneoplastic hepatocytes in the rat after administration of hepatocarcinogens or X-rays.

The urinary excretion of seven aldehydes, acetone, coproporphyrin III and 8-hydroxy-2′-deoxyguanosine (8-OH-dG) as non-invasive biomarkers of oxidative damage was measured in rats treated with diquat or N-nitrosodimethylamine (NDMA), two compounds causing hepatic damage by different mechanisms. Furthermore, the effect of co-administration of the aldehyde dehydrogenase inhibitor, calcium carbimide (CC) on the urinary excretion of the aldehydes was determined. Slight hepatotoxicity was found at the end of the experiment after treatment with NDMA (0.5, 4 and 8 mg/kg at t=0, 48 and 96 h, respectively) or diquat (6.8 and 13.6 mg/kg at t=0 and 48 h, respectively). In diquat treated rats slight nephrotoxicity was also found. Urinary excretion of aldehydes, acetone and coproporphyrin III remained largely unchanged in rats treated with NDMA. In the rats treated with diquat, the urinary excretion of several aldehydes was several-fold increased. An increase was also found in the urinary excretion of 8-OH-dG after the second dose of diquat. Treatment of rats with CC did not significantly influence the urinary excretion of aldehydes in control and NDMA rats. However, in rats treated with diquat, CC caused a potentiating effect on the excretion of acetaldehyde, hexanal and malondialdehyde (MDA), indicating that oxidation of aldehydes to carbonylic acids by aldehyde dehydrogenases (ALDHs) might be an important route of metabolism of aldehydes. In conclusion, increased urinary excretion of various aldehydes, acetone, coproporphyrin III and 8-OH-dG was observed after administration of diquat, probably reflecting oxidative damage induced by this compound. No such increases were found after NDMA administration, which is consistent with a different toxicity mechanism for NDMA. Therefore, excretion of aldehydes, acetone, coproporphyrin III and 8-OH-dG might be used as easily accessible urinary biomarkers of free radical damage.

Free radical damage is an important factor in many pathological and toxicological processes. Despite extensive research efforts in biomarkers in recent years, yielding promising results in experimental animals, there is still a great need for additional research on the applicability of, especially non-invasive, biomarkers of free radical damage in humans. This review gives an overview of the applications in experimental and human situations of four main groups of products resulting from free radical damage, these include: lipid peroxidation products, isoprostanes, DNA-hydroxylation products and protein hydroxylation products.

P53 protein plays an important role in regulation of the cell cycle. Recently, a role in tumour genesis has also been suggested. The protein is induced after various forms of DNA damage. Immunohistochemical detection of p53 protein showed positive cells in human skin after UV-irradiation, in mouse skin after benzo[a]pyrene treatment and in mouse spleen, thymus and bone after gamma-irradiation. However, no staining was found in mouse and rat liver with traditional immunohistochemical staining methods due to the low amount of p53 present. This seriously hampered studies on the role of p53 in hepatocarcinogenesis. We have developed a more sensitive immunohistochemical method for staining of p53 in paraffin-embedded sections of rat liver using microwave irradiation for antigen retrieval, avidin-biotin complexing and tyramide amplification. A strong, specific fluorescence signal for p53 was found in hepatocytes of rats that had received the hepatocarcinogen N-hydroxy-2-acetylaminofluorene; in control liver no such p53 staining was observed. The fluorescence was located in the nucleus of hepatocytes in zone 1 of the liver. This agrees with the fact that N-hydroxy-2-acetylaminofluorene causes cytotoxicity in this zone.

The flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) and its metabolite 2-bromoacrolein (2BA) are very potent bacterial mutagens in Salmonella typhimurium (S. typhimurium) TA 100. In this study, we showed that 2BA and Tris-BP are also mutagenic in S. typhimurium TA 104, which detects mutations at AT base pairs, while TA 100 detects mutations at CG basepairs. We also studied the mutagenicity of 2BA in mammalian cells in vitro and in the rat in vivo. Firstly, 2BA was tested in the human lymphoblastoid cell line TK6. The results showed that there was no increase in mutation frequency at the hprt locus, whereas there was a large decrease in cell survival. Secondly, a shuttle vector system was used to study the induction of mutations by 2BA:DNA adducts. The vector was modified by insertion of a single-stranded oligonucleotide containing on average one 2BA:DNA adduct. No increase in mutation frequency above background was detected after replication of this vector in SV40 transformed normal human fibroblasts. Because the liver is a major site for bioactivation of Tris-BP to 2BA in vivo, we tested the initiating capacity of Tris-BP in the rat liver in a modified Solt & Farber initiation and promotion system. Administration of Tris-BP resulted in a small increase in the number of preneoplastic gamma-glutamyl-transpeptidase positive (GGT+) foci in the liver compared to control animals (only significant in the lowest size class). Modification of the experimental protocol by performing partial hepatectomy 24 h after the administration of Tris-BP, did not increase the number of GGT+ or glutathione S-transferase-P (GST-P+) positive foci above the control level. Taken together, these results indicate that, in spite of a high mutagenicity in S. typhimurium, 2BA and Tris-BP have low or negligible mutagenic effects in mammalian systems. The lack of mutagenic activity may explain why Tris-BP is not a carcinogen in the rat liver.

Carbon tetrachloride (CCl4) is a model compound for inducing free radical damage in liver. In this study 10 biomarkers in rats treated ip with three different single doses of CCl4(0.25, 0.50, and 1.00 ml/kg body wt) were measured dose and time dependently and compared to evaluate these urinary products as noninvasive biomarkers for radical damage. Eight degradation products of lipid peroxides, namely, formaldehyde, acetaldehyde, acetone, propanal, butanal, pentanal, hexanal, and malondialdehyde (MDA), 8-hydroxy-2′-deoxyguanosine (8-OH-dG) and coproporphyrin III were measured in this study. As general measures of toxicity, several clinical chemical parameters (n= 12) and histopathological damage were determined. A dose-dependent increase in both the clinical parameters and the lipid degradation products was found. Increases in lipid degradation products were statistically significant at doses of 0.5 and 1 ml/kg CCl4. An increase in these products was already found in the first 12 h after exposure. At the lowest dose, 0.25 ml/kg CCl4, acetaldehyde and propanal already showed a statistically significant increase as well. No change in the urinary levels of 8-OH-dG could be found in this study and a decrease in the urinary excretion of coproporphyrin III was found. It is concluded that 8-OH-dG and coproporphyrin III are not useful biomarkers for radical damage induced by CCl4. Lipid degradation products, however, are promising noninvasive biomarkers forin vivoradical damage, although the precise specificity of these biomarkers for damage induced by radicals needs to be further investigated.