Pharmacokinetics and metabolism of benzophenone 2 in the rat.

Twelve derivatives of benzophenone (BP1-BP12) are widely used as UV-screens to protect industrial products from light induced damage. There is growing public concern about industrially produced chemicals that might interfere with hormonal signalling pathways, thus having potential adverse effects on human health. The derivative 2,2',4,4'-tetrahydroxybenzophenone (BP2) which is used in cosmetic products and in packaging materials, was previously shown to be an estrogenic endocrine active chemical (EAC). While the metabolisation of BP3 has been analyzed in vivo, according to our knowledge little is known about the pharmacokinetics of BP2. Therefore we performed a dose-response experiment with 5 dosages of BP2 which was applied per gavage to adult ovariectomised (ovx) rats for 5 days. Serum samples were analyzed via HPLC. Metabolites were further identified by Helix pomatia glucuronidase treatment and subsequent ion-trap-mass spectrometry. Additionally we analyzed the time dependent metabolisation and excretion of BP2 in a kinetic study. The parent compound BP2 is metabolised to glucuronide - and sulfate-conjugates. In the serum maximum levels of BP2, BP2-glucuronide and BP2-sulfate were observed already 30 min after BP2 application while highest concentrations of BP2 and its metabolites in urine were measured 120 min after treatment. It is suggested that this biotransformation occurs via a first-pass effect in the gut wall or the liver. Despite this rapid metabolisation and excretion, the amount of unconjugated BP2 was sufficient to induce a dose dependent estrogenic effect in the uterotrophic assay.

Intercellular communication of DNA damage and oxidative status underpin bystander effects.

PURPOSE: A well-known phenomenon in the field of radiation biology is that cells exposed to ionizing radiation (IR) (targeted cells) can induce in non-irradiated (non-targeted), bystander cells effects reminiscent of DNA damage responses (DDR) normally expected, exclusively in targeted cells. These phenomena are collectively referred to as radiation-induced bystander effects (RIBE) and have different manifestations depending on the endpoint studied. Although it is now recognized that RIBE reflects to a considerable extent communication by the targeted cells to undamaged cells of their damaged status, the molecular underpinnings of this communication and its significance for the organism are only partly understood. In particular, it remains unknown why and how targeted cells induce DNA damage in non-targeted, bystander cells threatening their genomic stability and risking thus their transformation to cancer cells. Here, we outline observations hinting to possible sources of artifacts in experiments designed to detect RIBE and summarize a model according to which targeted cells modulate their redox status as part of their overall response to IR and use this modified redox status as a source to generate signals that are transmitted to non-irradiated cells of the organism. MATERIAL AND METHODS: A synthesis of published evidence is presented. RESULTS: Depending on type, RIBE signals may be transmitted through various forms of direct intercellular contact, through molecules acting locally in a paracrine fashion, or through molecules acting remotely in an endocrine fashion. We reason that DNA damage generated in bystander cells is unlikely to manifest the clustered character exhibited in directly exposed cells and postulate that RIBE will depend on complications generated when simpler forms of damage encounter the DNA replication fork. CONCLUSIONS: We suggest that RIBE result from intercellular communication mechanisms designed to spread within tissues, or the organism, alarm signals of DNA damage inflicted in subsets of the constituent cells. This response likely evolved to protect organisms by appropriately modulating stress response, repair or apoptosis, and may in some instances also cause adverse effects, e.g. as collateral damage.

The ultraviolet filter benzophenone 2 interferes with the thyroid hormone axis in rats and is a potent in vitro inhibitor of human recombinant thyroid peroxidase.

Endocrine disrupting chemicals (EDCs), either plant constituents or contaminants deriving from industrial products, may interfere with the thyroid hormone (TH) axis. Here, we examined whether selected EDCs inhibit the key reactions of TH biosynthesis catalyzed by thyroid peroxidase (TPO). We used a novel in vitro assay based on human recombinant TPO (hrTPO) stably transfected into the human follicular thyroid carcinoma cell line FTC-238. F21388 (synthetic flavonoid), bisphenol A (building block for polycarbonates), and the UV filter benzophenone 2 (BP2) inhibited hrTPO. BP2 is contained in numerous cosmetics of daily use and may be in regular contact with human skin. Half-maximal inhibition in the guaiacol assay occurred at 450 nmol/liter BP2, a concentration 20- and 200-fold lower than those required in case of the TPO-inhibiting antithyroid drugs methimazole and propylthiouracil, respectively. BP2 at 300 nmol/liter combined with the TPO substrate H(2)O(2) (10 mumol/liter) inactivated hrTPO; this was, however, prevented by micromolar amounts of iodide. BP2 did not inhibit iodide uptake into FRTL-5 cells. In BP2-treated rats (333 and 1000 mg/kg body weight), serum total T(4) was significantly decreased and serum thyrotropin was significantly increased. TPO activities in the thyroids of treated animals were unchanged, a finding also described for methimazole and propylthiouracil. Thus, EDCs, most potently BP2, may disturb TH homeostasis by inhibiting or inactivating TPO, effects that are even more pronounced in the absence of iodide. This new challenge for endocrine regulation must be considered in the context of a still prevailing iodide deficiency in many parts of the world.

Effects of a 5-day treatment with the UV-filter octyl-methoxycinnamate (OMC) on the function of the hypothalamo-pituitary-thyroid function in rats.

Octyl-methoxycinnamate (OMC) is one of the most frequently used UV-filters in sunscreens to protect the skin against the noxious influence of UV radiation. Recently, OMC was suspected to act as an "endocrine active chemical" (EAC) with estrogenic actions. While EACs have been investigated thoroughly for interference with reproductive function in mammalians, surprisingly little efforts have been made to investigate an interference of EACs with the hypothalamo-pituitary-thyroid (HPT) axis despite the expression of estrogen receptors in all parts of this axis. Therefore, we conducted an in vivo study with ovariectomised rats treated for 5 days with different doses of OMC or 17beta-estradiol (E2) as a control. Determined parameters comprised serum levels of TSH, T4 and T3, hypothalamic TRH mRNA expression, protein-expression of the sodium-iodide-symporter (NIS) and the TSH receptor and the activities of thyroid peroxidase (TPO) in the thyroid and the T3-responsive hepatic type I 5'deiodinase (Dio1) in the liver. While E2 did not affect TSH-, T4- or T3-levels, OMC caused a dose-dependent decrease of serum concentrations of all of these hormones. TRH expression remained unaffected, while in the thyroid, expression of the TSH receptor but not of NIS was stimulated by OMC. TPO activity was unaltered but Dio1 activity was reduced by OMC. Thus, our results demonstrate a non-estrogenic interference of OMC within the rodent HPT axis with inadequate feedback response to impaired thyroid hormone status, indicated by decreased serum thyroid hormone and hepatic Dio1 levels.

Multi-organic risk assessment of estrogenic properties of octyl-methoxycinnamate in vivo A 5-day sub-acute pharmacodynamic study with ovariectomized rats.

Sun protection products contain a variety of UV-filters, among others, octyl-methoxycinnamate (OMC). Recently, an uterotrophic effect in immature rats has been reported, indicating that OMC might have estrogenic properties and thus is an endocrine active chemical (EAC). However, determination of an estrogenic activity in the uterus only is a restricted approach with the potential risk of missing undesirable actions in other organs regulated by estrogens. A pharmacodynamic experiment with 5 dosages of OMC in adult ovariectomized (ovx) rats was carried out to quantify the multi-organic estrogenic properties of OMC. As control compound, estradiol-valerate (E2) was included. Animals were treated per gavage for 5 days. The expression levels of markers of estrogenic action in several organs were measured by RT-PCR. Effects on metabolic parameters were assessed by determination of the serum concentrations of leptin, cholesterol, high and low density lipoproteins (HDL and LDL), glucose and triglycerides. Observed changes upon OMC treatment were analyzed using the NO(A)EL and the benchmark dose approach. From the obtained pharmacodynamic data of the most sensitive parameter (truncated estrogen receptor protein 1 gene expression in the pituitary) we obtained threshold values that are exceeded by the recommended use of OMC containing formulations for skin protection in humans, therefore we propose to reduce the use of OMC in cosmetic products. In addition to estrogenic actions of OMC, non-estrogenic effects have been found for this chemical supporting the need of a multi-organic risk assessment of putative EACs.

Bystander effects as manifestation of intercellular communication of DNA damage and of the cellular oxidative status.

It is becoming increasingly clear that cells exposed to ionizing radiation (IR) and other genotoxic agents (targeted cells) can communicate their DNA damage response (DDR) status to cells that have not been directly irradiated (bystander cells). The term radiation-induced bystander effects (RIBE) describes facets of this phenomenon, but its molecular underpinnings are incompletely characterized. Consequences of DDR in bystander cells have been extensively studied and include transformation and mutation induction; micronuclei, chromosome aberration and sister chromatid exchange formation; as well as modulations in gene expression, proliferation and differentiation patterns. A fundamental question arising from such observations is why targeted cells induce DNA damage in non-targeted, bystander cells threatening thus their genomic stability and risking the induction of cancer. Here, we review and synthesize available literature to gather support for a model according to which targeted cells modulate as part of DDR their redox status and use it as a source to generate signals for neighboring cells. Such signals can be either small molecules transported to adjacent non-targeted cells via gap-junction intercellular communication (GJIC), or secreted factors that can reach remote, non-targeted cells by diffusion or through the circulation. We review evidence that such signals can induce in the recipient cell modulations of redox status similar to those seen in the originating targeted cell - occasionally though self-amplifying feedback loops. The resulting increase of oxidative stress in bystander cells induces, often in conjunction with DNA replication, the observed DDR-like responses that are at times strong enough to cause apoptosis. We reason that RIBE reflect the function of intercellular communication mechanisms designed to spread within tissues, or the entire organism, information about DNA damage inflicted to individual, constituent cells. Such responses are thought to protect the organism by enhancing repair in a community of cells and by eliminating severely damaged cells.

Effects of estradiol, benzophenone-2 and benzophenone-3 on the expression pattern of the estrogen receptors (ER) alpha and beta, the estrogen receptor-related receptor 1 (ERR1) and the aryl hydrocarbon receptor (AhR) in adult ovariectomized rats.

The estrogen receptors (ERs) are members of a super family of ligand-activated transcription factors mediating estrogenic responses. A close functional kinship was found for the structurally related estrogen receptor-related receptor1 (ERR1), a constitutively active transcription factor. The aryl hydrocarbon receptor (AhR) mediates the toxic and estrogenic effects of a wide variety of environmental contaminants and industrial pollutants. Both the ERR1 and the AhR are known to modulate the ER's signalling pathways in multiple ways. Organic chemicals with a certain structural relationship to steroid hormones often induce a tissue- or cell-specific variety of responses distinct from estrogenic responses and this may involve ERR1 and AhR. The UV-screens benzophenone-2 and benzophenone-3 (BP2, BP3), structurally related to known steroid receptor ligands, are used in cosmetics and plastics to improve product stability and durability. Both BP2 and BP3 were shown to exert uterotrophic effects and BP2 was shown to bind to the estrogen receptors. Whether such effects are also exerted in other organs is unknown. Therefore, an approach to a multi-organic risk assessment for these substances was made by measuring the gene-expression of the four mentioned receptors in the pituitary, the uterus and the thyroid after a five-day treatment in comparison to estradiol. Though BP2 seems to exert an estrogen-like effect while BP3 does not, there are regulatory effects on receptor expression for both substances that indicate a kind of endocrine disruption that is not assessed by "classical" estrogenic markers.

The impact of serotonin on the development of bystander damage assessed by γ-H2AX foci analysis.

PURPOSE: This investigation addresses the question of whether serum constituents have a modulating effect on the induction of bystander effects. It was carried out as part of an inter-laboratory comparison-exercise within the European Union-integrated project NOTE (Non-targeted effects of ionizing radiation). Serotonin is a neurotransmitter, which is present in the circulatory system and therefore in blood serum. Fetal bovine serum is an essential supplement to most cell culture media and contains different, batch-dependent concentrations of serotonin. In a recent publication, serotonin was found to have a strong modulating effect on the size of bystander effects detected using colony formation assays. MATERIALS AND METHODS: We tested modulatory effects on bystander response detected by γ-H2AX (phosphorylated histone H2AX) foci formation in cells exposed to media of irradiated cells. Cells were grown in serum batches with different serotonin concentrations. RESULTS: While the data obtained with the three serum batches examined show a trend for increasing bystander response with increasing serotonin concentration, the effect failed to reach statistical significance. CONCLUSIONS: Serotonin may have a modulating effect on radiation-induced bystander effects, but more work is required to conclusively characterize this contribution.

Dependence of adaptive response and its bystander transmission on the genetic background of tested cells.

PURPOSE: Radiation-induced adaptive response (AR) is a phenomenon of increased radioresistance mediated by a low priming dose of ionizing radiation (IR) applied prior to a higher challenging dose. We have previously shown that in mouse-embryo fibroblasts (MEF) and human A549 cells, AR is associated with enhanced repair of DNA double-strand breaks (DSB) by the DNA-PK-dependent pathway of non-homologous end-joining (D-NHEJ). Importantly, AR was 'transmitted' to non-irradiated bystander cells through transfer of medium from cells that had received a priming dose of IR. Here, we examine the influence of the genetic background in these responses. MATERIALS AND METHODS: Two plasmid-based assays specifically designed to measure the efficiency of NHEJ and HRR (homologous recombination repair) were deployed. MEF and the primary human fibroblast cell lines HF12 and HF19 were exposed to 10 mGy to 5 Gy X-rays. Bystander effects were investigated using the medium-transfer technique. RESULTS: In contrast to MEF, which induce robust AR to NHEJ, even as a bystander response, human fibroblasts fail to develop such phenomena. CONCLUSIONS: The development of AR is cell-type-specific. The same holds true for the development of AR as a bystander effect. Better understanding of the underlying mechanisms will help to understand the molecular basis of these differences in response.

Widespread dependence of backup NHEJ on growth state: ramifications for the use of DNA-PK inhibitors.

PURPOSE: The backup pathway of nonhomologous end joining (B-NHEJ) enables cells to process DNA double-strand breaks (DSBs) when the DNA-PK-dependent pathway of NHEJ (D-NHEJ) is compromised. Our previous results show marked reduction in the activity of B-NHEJ when LIG4(-/-) mouse embryo fibroblasts (MEFs) cease to grow and enter a plateau phase. The dependence of B-NHEJ on growth state is substantially stronger than that of D-NHEJ and points to regulatory mechanisms or processing determinants that require elucidation. Because the different D-NHEJ mutants show phenotypes distinct in their details, it is necessary to characterize the dependence of their DSB repair capacity on growth state and to explore species-specific responses. METHODS AND MATERIALS: DSB repair was measured in cells of different genetic background from various species using pulsed-field gel electrophoresis, or the formation of γ-H2AX foci, at different stages of growth. RESULTS: Using pulsed-field gel electrophoresis, we report a marked reduction of B-NHEJ during the plateau phase of growth in KU and XRCC4, mouse or Chinese hamster, mutants. Notably, this reduction is only marginal in DNA-PKcs-deficient cells. However, reduced B-NHEJ is also observed in repair proficient, plateau-phase cells after treatment with DNA-PK inhibitors. The reduction of B-NHEJ activity in the plateau phase of growth does not derive from the reduced expression of participating proteins, is detectable by γ-H2AX foci analysis, and leads to enhanced cell killing. CONCLUSIONS: These results further document the marked dependence on growth state of an essential DSB repair pathway and show the general nature of the effect. Molecular characterization of the mechanism underlying this response will help to optimize the administration of DNA repair inhibitors as adjuvants in radiation therapy.

Evidence of an adaptive response targeting DNA nonhomologous end joining and its transmission to bystander cells.

Adaptive response (AR) is a term describing resistance to ionizing radiation-induced killing or formation of aberrant chromosomes that is mediated by pre-exposure to low ionizing radiation doses. The mechanism of AR remains elusive. Because cell killing and chromosome aberration formation derive from erroneous processing of DNA double-strand breaks (DSB), AR may reflect a modulation of DSB processing by nonhomologous end joining (NHEJ) or homologous recombination repair. Here, we use plasmid end-joining assays to quantify modulations induced by low ionizing radiation doses to NHEJ, the dominant pathway of DSB repair in higher eukaryotes, and investigate propagation of this response through medium transfer to nonirradiated bystander cells. Mouse embryo fibroblasts were conditioned with 10 to 1000 mGy and NHEJ quantified at different times thereafter by challenging with reporter plasmids containing a DSB. We show robust increases in NHEJ efficiency in mouse embryo fibroblasts exposed to ionizing radiation >100 mGy, irrespective of reporter plasmid used. Human tumor cells also show AR of similar magnitude that is compromised by caffeine, an inhibitor of DNA damage signaling acting by inhibiting ATM, ATR, and DNA-PKcs. Growth medium from pre-irradiated cells induces a caffeine-sensitive AR in nonirradiated cells, similar in magnitude to that seen in irradiated cells. In bystander cells, γH2AX foci are specifically detected in late S-G(2) phase and are associated with Rad51 foci that signify the function of homologous recombination repair, possibly on DNA replication-mediated DSBs. The results point to enhanced NHEJ as a mechanism of AR and suggest that AR may be transmitted to bystander cells through factors generating replication-mediated DSBs.

A dose-response study on the estrogenic activity of benzophenone-2 on various endpoints in the serum, pituitary and uterus of female rats.

The tetrahydroxylated biphenyl-ketone 2,2',4,4'-tetrahydroxybenzophenone (BP2), one of twelve benzophenone-derived UV-filters, is used in cosmetic products and in packaging materials to protect these products from light induced damage. Recently published studies showed that BP2 exerts estrogenic activity; thus, it is an endocrine active chemical. We present data from a pharmacodynamic dose-response experiment with five dosages of BP2 applied per gavage to adult ovariectomized (ovx) rats for 5 days. Estradiol-valerate (E2) served as a control compound. The uterotrophic assay, proposed by the OECD, was modified to have a broader view on endocrine activity outside the urogenital tract to prevent that undesirable actions in other organs regulated by estrogens are missed. The gene expression levels of marker genes of estrogenic action were measured by semi-quantitative RT-PCR. Metabolic parameters were assessed by determination of the serum concentrations of leptin, cholesterol, high- and low-density lipoproteins, and triglycerides in the serum. Administration of BP2 at dosages of 10-1,000 mg/kg bodyweight led to changes of these parameters comparable to the changes in the E2 group with 0.6 mg/kg bodyweight. For the observed estrogenic activities of BP2, the "no observed adverse effect levels" were determined. Additionally, the data were further analyzed using the benchmark approach. If BP2 is transcutaneously absorbed in the human, the obtained threshold values would suggest refraining from the further use of BP2 as UV-filter in cosmetic products although additional toxicological studies should be conducted to clarify possible adverse effects.