Cytotoxic effects of BADGE (bisphenol A diglycidyl ether) and BFDGE (bisphenol F diglycidyl ether) on Caco-2 cells in vitro

Bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE) are used as starting substances for the manufacturing of epoxy resins used in internal can coatings. They are obtained by a condensation reaction between epichlorohydrin with bisphenol A and bisphenol F, respectively. These potential endocrine disrupting chemicals are able to enter the food chain and to reach the intestinal epithelium, causing structural and functional damages. The human colorectal adenocarcinoma cell line Caco-2 is a widely used in vitro model of the intestinal cells. The aim of this study was to characterize BADGE and BFDGE toxicity in Caco-2 cells, in particular, at the cellular and molecular level. Using several approaches, we characterized BADGE- and BFDGE-induced cell toxicity in Caco-2 cells. The treatment was done using different concentrations up to cytotoxic doses and different times of exposure to the agents. We evaluated the effect of these compounds on cell morphology, cell detachment, cell proliferation, F-actin disruption and plasma membrane integrity. Both compounds are able to induce morphological changes, cell detachment from the substratum and to inhibit cell proliferation, being these effects time and dose-dependent. Moreover, BADGE and BFDGE induce F-actin depolymerization, this effect is very potent at 24 h of incubation with the agents and a complete F-actin disruption can be observed at 200 μM BADGE or BFDGE. In addition, cell integrity is not damaged, since neither propidium iodide uptake nor LDH release takes place in Caco-2 cells exposed to high doses of these agents for 24 h.     

Determination of bisphenol diglycidyl ethers in topical dosage forms

A method involving extraction and LC-ESI-MS-MS detection of BADGE, BFDGE, BADGE*H2O, BADGE*2H2O, BADGE*HCl, BADGE*H2O*HCl, BADGE.2HCl and BFDGE*2HCl in aqueous cream was developed and validated. Initially, empty internally lacquered aluminum container closure systems were extracted with isopropanol as an attempt to estimate the upper limit of extractable bisphenol diglycidyl ethers present in lacquer. Six of the eight potential bisphenol diglycidyl ethers were quantified. In an accelerated experiment, on aqueous cream stored in lacquered aluminum tubes at 70 degrees C, all derivatives except BADGE*2HCl and BFDGE*2HCl were extracted from cream samples and quantified as an attempt to estimate the upper limit of compounds leaching to the cream. Detection limits were from 0.3+/-0.2 to 3.4+/-0.7 microgl(-1). Recoveries were determined for all compounds at three concentration levels (mean 63+/-6%). Mean inter-day and mean intra-day precision was 7+/-2 and 13+/-6%, respectively. Three commercially available creams were obtained from a local community pharmacy and analysed for bisphenol diglycidyl ethers. BADGE, BADGE*H2O, BADGE*2H2O and BADGE*H2O*HCl were detected and quantified. In conclusion, the developed method allows for the extraction and detection of bisphenol diglycidyl ethers originating from the epoxy phenol lacquer used in aluminum tubes. This study does not indicate that they leach into aqueous cream in significant amounts under normal storage conditions.     

In vitro assay of hydrolysis and chlorohydroxy derivatives of bisphenol A diglycidyl ether for estrogenic activity

Bisphenol A diglycidyl ether (BADGE) is an epoxy resin monomer. Epoxy-based solution coatings are used in many applications as additives for a variety of plastic coatings in food packaging. It is well known that unreacted BADGE can migrate from epoxy-based packing materials into foods. Not only BADGE but also its derivatives can easily migrate into foods and it is likely that we intake BADGE and its derivatives through food or drink. Recently, endocrine disrupting chemicals (EDCs) have attracted attention because they have been shown to affect reproduction in wildlife. The estrogenic activity of BADGE derivatives has not previously been investigated. Therefore, we investigated the estrogenic activity of the BADGE derivatives, dihydrolysed BADGE (BADGE-4OH) and chlorohydroxy BADGE (BADGE-2Cl), using breast cancer cell (T47D) proliferation assay and estrogen receptor (ER) () binding assay. These chemicals exhibited T47D cell proliferation at concentrations of 10⁻¹⁴–10⁻⁴ . However, these chemicals did not bind to ER () in the binding assay.     

Steroid activities comparison of natural and food wrap compounds in human breast cancer cell lines.

In this study, we tested and compared the endocrine disruption activities of compounds in materials used to package foods (bisphenol A, bisphenol F, and bisphenol A diglycidylether BADGE) with natural molecules (genistein, apigenin, kaempferol, and tangeretin) in the human breast cancer cell lines MCF-7 (ER(+)) and MDA-MB453 (AR(+); GR(+)). Octylphenol was also chosen as a xenoestrogen reference. Two compounds had no estrogenic activity: BADGE and tangeretin. Genistein was the most active compound in the E-Screen assay with MCF-7, followed by octylphenol, bisphenol F, bisphenol A and apigenin, with kaempferol the least potent. All estrogenic compounds competed with 17beta-estradiol for binding to the MCF-7 ER and their estrogenic effects were abolished in the presence of tamoxifen, an ER antagonist. In MDA-MB453 cells transfected with pMMTVneo-Luc, all compounds had anti-androgenic activities, with octylphenol the most potent. Kaempferol, genistein, and apigenin were more potent anti-androgens than bisphenols A or F. The natural compounds had a biphasic effect on luciferase activity. At high concentrations, genistein (10(-5)M) and apigenin (10(-6)M) acted as GR agonists in transfected MDA-MB453 cells. Furthermore, apigenin, at a concentration of 10(-5)M, may act as a partial androgen receptor (AR) agonist, as nilutamide, an AR antagonist, inhibited its activity by 26%.     

Migration of BADGE (bisphenol A diglycidyl-ether) and BFDGE (bisphenol F diglycidyl-ether) in canned seafood

Migration of potentially toxic materials used for the lining of commercial can goods remains an important issue, especially with respect to certain types of processed foods. Seafood is one type where more information is needed with respect to other ingredients used for adding value to fishery products. Most cans are internally coated with starters of resins such as bisphenol A diglycidyl-ether (BADGE) and bisphenol F diglycidyl-ether (BFDGE), both considered as toxic compounds. Several seafood products, sardines, tuna fish, mackerel, mussels, cod and mackerel eggs, were manufactured in different conditions changing covering sauce, time and temperature of storage and heat-treated for sterilization in cans. Migration kinetics of BADGE and BFDGE from varnish into canned products were evaluated by HPLC in 70 samples after 6, 12 or 18 months of storage. Results showed that there is no migration of BADGE in tuna fish, sardines, mussels or cod. However, migration of BFDGE occurs in all species, in a storage time-dependent way and content of fat, although migration of these compounds is not affected by sterilization conditions. All samples analyzed presented values lower than 9 mg BADGE/kg net product without exceeding European limits. However, concerning BFDGE migration, European legislation does not allow the use and/or the presence of BFDGE. Main migration takes place in mackerel reaching the highest values, 0.74 mg BFDGE/kg and 0.34 mg BADGE/kg net product, in red pepper sauce.     

Genotoxic and endocrine activities of bis(hydroxyphenyl)methane (bisphenol F) and its derivatives in the HepG2 cell line

Human can be exposed to bis(hydroxyphenyl)methane (bisphenol F or BPF) and its derivatives as environment and food's contaminants. This study was investigated to identify and to compare toxic potency of BPF, BFDGE, and two of BPF metabolites using in vitro methods. BPF did not induce any genic mutation in bacteria when the Ames test was performed according to the OECD guideline. In contrast, using Human cell lines and Comet assay, we demonstrated that BPF and Bisphenol F Diglycidyl Ether (BFDGE) were effective on HepG2 cell DNA fragmentation at non-cytotoxic concentrations. DHB was also positive but at higher concentrations, near its limit of solubility. Neither BPF, nor DHB induced a positive response in the micronucleus assay. The increase of micronuclei observed when cells were exposed to BFDGE was mostly due to a cytotoxic effect. Concerning endocrine activities, BPF increased the luciferase activity in HepG2 cells transiently transfected with a concentration dependant pattern, DHB also induced a positive response but at highest concentrations. Estrogenic responses in the HepG2 cells differed with the estrogen receptor (ER) involved. Using MDA-kb2 cell line stably transfected with pMMTV-neo-Luc, only BPF was anti-androgenic at the highest concentration (10(-5)M). Then, we demonstrated using human cell lines, especially HepG2, BPF was the most toxic compound in term of genotoxicity and endocrine activities compared to DHB and BPF-OH, the free metabolites identified in rat urine when BPF was administrated to rats.     

Estrogenic and anti-androgenic activity of nitrophenols in diesel exhaust particles (DEP)

We recently isolated 4-nitrophenol, 2-methyl-4-nitrophenol, 3-methyl-4-nitrophenol, and 4-nitro-3-phenylphenol from diesel exhaust particles (DEP) and identified them as vasodilators. Because these compounds are alkylphenolic derivatives that might mimic hormones, we evaluated their estrogenic activity by human estrogen receptor (hER)-yeast screen assay. All of these nitrophenol derivatives except 2-methyl-4-nitrophenol exhibited estrogenic activity. Some estrogenic compounds are also anti-androgenic, so we measured the anti-androgenic activity of the same compounds by human androgen receptor (hAR)-yeast screen assay. We found anti-androgenicity in all four nitrophenols. Nitrophenols in DEP possess not only vasodilatory activity but also estrogenic and anti-androgenic activity.     

No androgenic/anti-androgenic effects of bisphenol-A in Hershberger assay using immature castrated rats

Several studies have demonstrated that bisphenol A (BPA) exhibited weak estrogenic activity in the 3-day uterotrophic assay using ovariectomized (OVX) and immature rats (Toxicol. Lett. 115 (2000) 231; Regul. Toxicol. Pharmacol. 32 (2000) 118; J. Toxicol. Sci. 26 (2001) 111) and BPA also possessed anti-androgenic activity in in vitro yeast based assays (J. Endocrinol. 158 (1998) 327). To investigate anti-androgenic effects of BPA. a rodent Hershberger assay was carried out using immature Sprague-Dawley male rats. An androgen agonist, testosterone (0.4 mg/kg per day), was administered for 7 consecutive days by subcutaneous (s.c.) injection as a positive control. Additionally, a pure androgen antagonist, flutamide (1, 5. 10 mg/kg per day. oral) was co-administered with testosterone (0.4 mg/kg per day s.c.). BPA was also administered orally with or without testosterone (0.4 mg/kg per day, s.c.) for 7 consecutive days. In the testosterone treated groups, glans penis, seminal vesicles, ventral prostate, and levator ani plus bulbocavernosus muscles (LABC) weights were significantly increased compared with control. However. flulamide dose-dependently inhibited the testosterone-induced re-growth of seminal vesicles, ventral prostate, and LABC, with a significant decrease at flutamide 1.0 mg/kg and above (P<0.05). Serum LH levels were also significantly increased (5 mg/kg and above, P<0.05), but no changes in serum testosterone levels. In contrast, BPA had no effects on the re-growth of seminal vesicles, ventral prostate and LABC induced by testosterone, and no significant differences were observed in serum LH and testosterone levels. In summary, the Hershberger assay could be a sensitive method for detecting androgenic or anti-androgenic chemicals, but BPA did not exhibit any androgenic or anti-androgenic activities in Hershberger assay.     

Comparative study of the endocrine-disrupting activity of bisphenol A and 19 related compounds.

The endocrine-disrupting activities of bisphenol A (BPA) and 19 related compounds were comparatively examined by means of different in vitro and in vivo reporter assays. BPA and some related compounds exhibited estrogenic activity in human breast cancer cell line MCF-7, but there were remarkable differences in activity. Tetrachlorobisphenol A (TCBPA) showed the highest activity, followed by bisphenol B, BPA, and tetramethylbisphenol A (TMBPA); 2,2-bis(4-hydroxyphenyl)-1-propanol, 1,1-bis(4-hydroxyphenyl)propionic acid and 2,2-diphenylpropane showed little or no activity. Anti-estrogenic activity against 17beta-estradiol was observed with TMBPA and tetrabromobisphenol A (TBBPA). TCBPA, TBBPA, and BPA gave positive responses in the in vivo uterotrophic assay using ovariectomized mice. In contrast, BPA and some related compounds showed significant inhibitory effects on the androgenic activity of 5alpha-dihydrotestosterone in mouse fibroblast cell line NIH3T3. TMBPA showed the highest antagonistic activity, followed by bisphenol AF, bisphenol AD, bisphenol B, and BPA. However, TBBPA, TCBPA, and 2,2-diphenylpropane were inactive. TBBPA, TCBPA, TMBPA, and 3,3'-dimethylbisphenol A exhibited significant thyroid hormonal activity towards rat pituitary cell line GH3, which releases growth hormone in a thyroid hormone-dependent manner. However, BPA and other derivatives did not show such activity. The results suggest that the 4-hydroxyl group of the A-phenyl ring and the B-phenyl ring of BPA derivatives are required for these hormonal activities, and substituents at the 3,5-positions of the phenyl rings and the bridging alkyl moiety markedly influence the activities.     

Possible androgenic/anti-androgenic activity of the insecticide fenitrothion

To date, within the field of endocrine disruption, much focus has been placed on chemicals that mimic oestrogens (so-called xenoestrogens), and the number of such chemicals apparently detected continues to grow steadily. Less effort has been expended on investigating chemicals that mimic, or antagonize, other hormones. Nevertheless, a number of chemicals have been reported to have a weak affinity for the androgen receptor, all of which have, to date, been found to have anti-androgenic activity in vivo. In this report, we present evidence that the insecticide fenitrothion can interact with the androgen, but not with the oestrogen, receptor. Using recombinant yeast expressing the human androgen receptor, we found that fenitrothion behaved as an androgen agonist in vitro when tested alone, and that it could antagonize the androgen DHT when both chemicals competed for the androgen receptor in vitro. In vivo studies using both intact and castrated male rats showed no conclusive androgenic or anti-androgenic responses. Changes in organ weights suggestive of anti-androgenic effects were mitigated against by the reduced body weights of fenitrothion-treated rats. The toxicity of the compound precluded the use of higher dose levels to substantiate any tentative findings. Interestingly, fenitrothion (and related insecticides) is structurally similar to flutamide, an anti-androgen used clinically that gives clearly positive responses in both intact and castrated rats.     

Scrutinizing the interactions between bisphenol analogues and plasma proteins: Insights from biomimetic liquid chromatography,molecular docking simulations and in silico predictions

The interactions between human serum albumin (HSA) and α₁- acid glycoprotein (AGP), the main plasma proteins binding drugs/xenobiotics, and some endocrine disrupting chemicals (EDCs), such as bisphenol A (BPA) and some of its structural analogues, bisphenol S (BPS), bisphenol F (BPF), bisphenol E (BPE), bisphenol B (BPB), bisphenol AF (BPAF), bisphenol A diglycidyl ether (BADGE) and bisphenol M (BPM), were characterized by biomimetic liquid chromatography (LC). The interactions between bisphenols (BPs) and either HSA or AGP protein was found to be non-specific and essentially lipophilicity-driven. To get more information on the binding of BPs and plasma proteins, in silico predictions and molecular docking simulations were exploited, and the results achieved in silico were compared to those observed in vitro. BPM was the one exhibiting the highest affinity on both plasma proteins according to these data. Our findings clarified the binding of these EDCs to plasma proteins and offered insights into the biodistribution and bioaccumulation processes underlying their toxicity.     

Re-evaluation of the first synthetic estrogen, 1-keto-1,2,3, 4-tetrahydrophenanthrene, and bisphenol A, using both the ovariectomised rat model used in 1933 and additional assays

1-Keto-1,2,3,4-tetrahydrophenanthrene (THP-1) was reported by Cook et al. in 1933 as the first synthetic estrogen. Estrogenic activity was assessed by the induction of vaginal cornification in ovariectomised rats. The corresponding 4-isomer (THP-4) was shown to be inactive. Both chemicals have been re-synthesised and assessed for hormonal activity. Each chemical bound weakly and to the same extent to isolated estrogen receptors, but only at high concentrations. However, they each lacked estrogenic or anti-estrogenic activity when evaluated in vitro using a yeast hER assay, and both failed to induce vaginal cornification or uterotrophic effects in ovariectomised rats. THP-1, and to a lesser extent THP-4, were shown to possess weak androgenic and anti-androgenic activity in vitro when evaluated using an hAR yeast assay. Estrogenic activity for bisphenol A (BPA) was subsequently demonstrated by [Dodds and Lawson, Synthetic, oestrogenic agents without the phenanthrene nucleus, Nature 137, (1936)] using the same ovariectomised rat protocol, and this activity has been confirmed and supplemented by positive uterotrophic effects for BPA in the same bioassays. The present results illustrate the complexity of deriving conclusions regarding the hormonal activities of chemicals. First, some activities observed in isolated hormonal receptor binding assays may not be expressed in functional hormonal assays. This indicates the need for functional hormonal assays in any screening programme. Second, that activities observed for a chemical in one hormonal assay may not be reflected in related hormonal assays. This indicates the need to define assay protocols with some precision when incorporating them into screening batteries. Finally, that some literature reports of hormonal activity for chemicals may not be capable of independent confirmation under apparently identical conditions of test. This illustrates the need to use lists of hormonally active chemicals with care.     

Effects of bisphenol A-related diphenylalkanes on vitellogenin production in male carp (Cyprinus carpio) hepatocytes and aromatase (CYP19) activity in human H295R adrenocortical carcinoma cells

The present study investigated the effects of the known xenoestrogen bisphenol A (BPA) relative to eight BPA-related diphenylalkanes on estrogen receptor (ER)-mediated vitellogenin (vtg) production in hepatocytes from male carp (Cyprinus carpio), and on aromatase (CYP19) activity in the human adrenocortical H295R carcinoma cell line. Of the eight diphenylalkanes, only 4,4'-(hexafluoropropylidene)diphenol (BHF) and 2,2'-bis(4-hydroxy-3-methylphenyl)propane (BPRO) induced vtg, i.e., to a maximum of 3% to 4% (at 100 microM) compared with 8% for BPA relative to the maximum induction by 17beta-estradiol (E2, 1 microM). Bisphenol A diglycidyl ether (BADGE) was a potent antagonist of vtg production with an IC50 of 5.5 microM, virtually 100% inhibition of vtg at 20 microM, and an inhibitive (IC50) potency about one-tenth that of the known ER antagonist tamoxifen (IC50, 0.6 microM). 2,2'-Diallyl bisphenol A, 4,4'-(1,4-phenylene-diisopropylidene)bisphenol, BPRO, and BHF were much less inhibitory with IC50 concentrations of 20-70 microM, and relative potencies of 0.03 and 0.009 with tamoxifen. Bisphenol ethoxylate showed no anti-estrogenicity (up to 100 microM), and 4,4'-isopropylidene-diphenol diacetate was only antagonistic at 100 microM. When comparing the (anti)estrogenic potencies of these bisphenol A analogues/diphenylalkanes, anti-estrogenicity occurred at lower concentrations than estrogenicity. 4,4'-Isopropylidenebis(2,6-dimethylphenol) (IC50, 2.0 microM) reduced E2-induced (EC50, 100 nM) vtg production due to concentration-dependent cytotoxicity as indicated by a parallel decrease in MTT activity and vtg, whereas the remaining diphenylalkanes did not cause any cytotoxicity relative to controls. None of the diphenylalkanes (up to 100 microM) induced EROD activity indicating that concentration-dependent, CYP1A enzyme-mediated metabolism of E2, or any Ah-receptor-mediated interaction with the ER, was not a likely explanation for the observed anti-estrogenic effects. At concentrations as great as 100 microM, none of the diphenylalkanes directly inhibited aromatase (CYP19) activity in H295R cells. Environmental exposure of fish to BPA and related diphenylalkanes, depending on the structure, may pose anti-estrogenic, and to a lesser extent estrogenic, risks to development and reproduction.     

Bisphenol A diglycidyl ether-induced apoptosis involves Bax/Bid-dependent mitochondrial release of apoptosis-inducing factor (AIF), cytochrome c and Smac/DIABLO

(1) Bisphenol A diglycidyl ether (BADGE) is a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) antagonist, which is able to induce apoptosis in tumor cells independently of PPAR-gamma in caspase-dependent and -independent manners. Additionally, BADGE promotes TRAIL-induced apoptosis. (2) We report that BADGE activates via Bax and caspases-2 and -8 both the intrinsic and extrinsic apoptotic pathways using Bid as a shunt. (3) BADGE stimulates the mitochondrial release of apoptosis-inducing factor (AIF), cytochrome c and second mitochondria-derived activator of caspase/direct IAP-binding protein with low pl (Smac/DIABLO). The release of cytochrome c could not be blocked by inhibitors of caspases-3, -8 and -9 indicating that BADGE acts upstream of caspases-3 and -9 and does not involve caspase-8 to release cytochrome c. (4) While the caspase-independent apoptotic effect might be mediated by AIF, the sensitizing effect of BADGE against other apoptotic substances is most likely mediated by the X-linked inhibitor of apoptosis inhibitor Smac/DIABLO. (5) Our data suggest that BADGE or BADGE derivatives could represent promising substances for the treatment of neoplasms improving the antitumoral activity of TRAIL.     

Estrogenic activity of alkylphenols, bisphenol S, and their chlorinated derivatives using a GFP expression system

Alkylphenol ethoxylates, widely used non-ionic surfactants, are biodegraded into alkylphenols such as nonylphenol (NP) and t-octylphenol (OP), short-chain ethoxylates such as NP-monoethoxylate (NP1EO) and NP-diethoxylate (NP2EO), and alkylphenoxy carboxylic acids such as 4-t-octylphenoxyacetic acid (OP1EC). Bisphenol S (BPS) is more heat-stable and photo-resistant than bisphenol A (BPA), and therefore replaces BPA. These chemicals could be chlorinated during wastewater treatment. We synthesized these compounds and their chlorinated derivatives to estimate their estrogenic activities using a GFP expression system. The EC(50) ranking of NP-related compounds was NP > ClNP > diClNP > NP1EO > ClNP1EO > NP2EO. The estrogenic activity of OP1EC was 10 times less potent than parent OP. Furthermore, BPS showed comparable estrogenic activity with BPA. The EC(50) ranking of BPS-related compounds was BPA ≥ BPS > triClBPS > diClBPS > ClBPS. Other tested BPS derivatives had no estrogenic activity. Chlorination of the tested chemicals did not enhance their estrogenic activity, in contrast to certain chlorinated BPAs. Thus, our results demonstrated that chlorinated derivatives of NP, OP, and BPS, even if artificially produced during wastewater processing, were less estrogenic than their parent chemicals, known as endocrine disruptors.     

Preliminary study of responses in mussel (Mytilus edilus) exposed to bisphenol A, diallyl phthalate and tetrabromodiphenyl ether

Environmental pollutants with hormonal activity including bisphenol, diallyl phtalate and tetrabromodiphenyl ether, have the potential to alter gonadal development and reproduction in aquatic wildlife. Little is known about the biological impact of environmentally relevant concentrations in mussels. To investigate some aspects of their potential estrogenic action, mussels were continuously exposed during 3 weeks. Gonadal development and vitellogenin like protein levels were examined. Bisphenol (50 microg/l) induced the expression of phospho-proteins in females and spawning in both sexes. Diallyl phthalate and tetrabromodiphenyl ether decreased phospho-protein levels in both sexes and induced spawning in males. Moreover, severe damaging effects on ovarian follicles and ovocytes were observed in both bisphenol A- and tetrabromodiphenyl ether-exposed female mussels.