Analysis of black fungal biofilms occurring at domestic water taps. I: compositional analysis using Tag-Encoded FLX Amplicon Pyrosequencing.

Mass growth of dark fungal biofilms on water taps and associated habitats was observed in various German drinking water distribution systems recently. Customers of affected drinking water systems are anxious about potential and unknown health risks. These environments are known to harbour a fungal flora also comprising a variety of fungal opportunists that are well known to cause superficial mycoses in humans (Exophiala equina, Exophiala lecanii-corni) but are not known to establish dark biofilms so far. To gain profound insight on composition of respective biofilms, a metagenomic approach using Tag-Encoded FLX Amplicon Pyrosequencing (TEFAP) of the ribosomal internal transcribed spacer 2 region in comparison with a classical cultivation approach using Sabouraud agar with chloramphenicol and erythritol-chloramphenicol-agar was performed. E. lecanii-corni was found to be the major component in 10 of 13 biofilms analysed independently of the method used. Alternaria sp., E. equina, Fusarium spp. and Ochroconis spp. were also relatively abundant. As expected, TEFAP usually revealed a higher diversity than the cultivation approaches. For example, opportunistic species like Candida albicans or Exophiala dermatitidis were detected in very low amounts. In conclusion, TEFAP turned out to be a promising and powerful tool for the semi-quantitative analysis of fungal biofilms. Referring to relevant literature, potential biological hazards caused by fungi of the dark biofilms can be regarded as low.

Analysis of black fungal biofilms occurring at domestic water taps. II: potential routes of entry.

Formation of tenacious and massive black biofilms was occasionally observed at the water-air interphase of water taps and in associated habitats at several locations in Germany. Exophiala lecanii-corni was proven to be the dominant component of these biofilms. Water utility companies were interested to understand by which route fungi building these black biofilms enter their habitat at affected sites in domestic sanitary. A wide variety of fungi is known to be common in wet indoor environments, as well as in the drinking water resources. Two possible routes of entry are therefore considered as follows: (a) distribution by the drinking water system or (b) a retrograde route of colonisation. Previous compositional analysis revealed that the black constituents of biofilms primarily belong to the herpotrichiellaceous black yeast and relatives. Therefore, a systematic search for black fungi in the drinking water system was performed using Sabouraud's glucose agar medium with chloramphenicol and erythritol-chloramphenicol agar as isolation media. Cadophora malorum was the dominant fungus in the investigated drinking water systems, and samples taken from the house connections (n = 50; 74 %, <200 cfu/L), followed by a so far undescribed Alternaria sp. (28 %; <10 cfu/L) and E. castellanii (26 %; <10 cfu/L). Of note, C. malorum was not present in any previously analysed biofilm. Since E. lecanii-corni was not found in any water sample from the distribution system tested, but represented the most abundant species in dark biofilms previously analysed, a retrograde route of contamination in case of E. lecanii-corni can be assumed.

Effects of boundary conditions on the cleaning efficiency of riverbank filtration and artificial groundwater recharge systems regarding bulk parameters and trace pollutants.

Drinking water is often produced from surface water by riverbank filtration (RBF) or artificial groundwater recharge (AGR). In this study, an AGR system was exemplarily investigated and results were compared with those of RBF systems, in which the effects of redox milieu, temperature and surface water discharge on the cleaning efficiency were evaluated. Besides bulk parameters such as DOC (dissolved organic carbon), organic trace pollutants including iodinated X-ray contrast media, personal care products, complexing agents, and pharmaceuticals were investigated. At all studied sites, levels of TOC (total organic carbon), DOC, AOX (adsorbable organic halides), SAC (spectral absorption coefficient at 254 nm), and turbidity were reduced significantly. DOC removal was stimulated at higher groundwater temperatures during AGR. Several substances were generally easily removable during both AGR and RBF, regardless of the site, season, discharge or redox regime. For some more refractory substances, however, removal efficiency turned out to be significantly influenced by redox conditions.

Kinetics and mechanisms of N-nitrosodimethylamine formation upon ozonation of N,N-dimethylsulfamide-containing waters: bromide catalysis.

N,N-Dimethylsulfamide (DMS), a newly identified, ubiquitous degradation product of the fungicide tolylfluanide, has been shown to be a N-nitrosodimethylamine (NDMA) precursor during ozonation. In this study, batch ozonation experiments in ultrapure buffered water, surface water, and tap water were performed to determine the kinetics and elucidate the mechanism of NDMA formation from DMS. It was found that at circumneutral pH, DMS reacts slowly with ozone (k approximately 20 M(-1) s(-1)) and moderately with hydroxyl radicals (k=1.5x10(9) M(-1)s(-1)). The reaction of DMS with these oxidants does not lead to NDMA. NDMA was only formed if bromide was present during ozonation of DMS-containing waters. Bromide is oxidized to hypobromous acid (HOBr) by ozone which then reacts with the primary amine of DMS to form a Br-DMS species. The rate limiting step of the formation of Br-DMS is the formation of HOBr. The reaction to form Br-DMS has an apparent second order rate constant at pH 8 of >3x10(4) M(-1)s(-1). The Br-DMS is transformed by ozone to NDMA and nitrate (k>or=5000 M(-1) s(-1)), with yields of 54% and 39%, respectively, based on the primary amine nitrogen of DMS. These reactions release bromide, making bromide a catalyst. NDMA is also formed during ozonation of DMS in the presence of hypochlorous acid (20-30% yield). The last step of NDMA formation is an intramolecular rearrangement with sulfur dioxide extrusion. On the basis of the mechanistic and kinetic information, it was possible to model NDMA formation in DMS-containing Lake Zurich water.

Approach for analytical characterization and toxicological assessment of ozonation products in drinking water on the example of acesulfame.

The monitoring and control of drinking water quality is generally important as it significantly contributes to the health of the population. In this context, particular attention has to be paid to the use of treatment techniques during drinking water treatment. It is known that the formation of reaction products (transformation products) has to be taken into account when oxidizing agents such as ozone are used. Different transformation products are classified as critical to health and require analytical examination. The risk assessment for previously unknown transformation products can be difficult as far as not all transformation products are present as single substances or the individual substances are not present in a sufficient high concentration or cannot be isolated from the original solution. The aim of this work is to show exemplarily the identification and quantification of ozonation products (OPs) after ozonation and their toxicological characterization, using the artificial sweetener acesulfame. It was shown that OPs can be fully characterized using ion chromatography in combination with different detection systems. A major OP could be recovered as a pure substance by crystallization and direct genotoxicological testing was possible without previous enrichment processes. Acesulfame samples of different concentrations in ultrapure and in drinking water after ozonation were tested in several genotoxicity tests. These tests revealed genotoxic effects of acesulfame after ozonation in ultrapure water in several genotoxicological test systems (micronucleus test, umu test, Ames-fluctuation-test and comet assay). In contrast, the crystallized ozonation product OP168 did not show any positive effects. Therefore, it seems likely that the observed effect was caused by the second major product OP170. However, a sufficiently large amount of analytically pure substance OP170 could not be obtained. It was also shown that the rate of the OP170 formation in drinking water is significantly lower than in ultrapure water and that ozonation in drinking water did not induce genotoxic effects.

Comparison of in vitro test systems using bacterial and mammalian cells for genotoxicity assessment within the "health-related indication value (HRIV) concept.

In numerous cases, the German health-related indication value (HRIV) concept has proved its practicability for the assessment of drinking water relevant trace substances (Umweltbundesamt 2003). The HRIV is based on the toxicological profile of a substance. An open point of the HRIV concept has been the assignment of standardized test procedures to be used for the assessment. The level of the HRIV is at its lowest as soon as the genotoxicity of the substance is detected. As a single test on its own, it is not sufficient enough to assess the human toxicological relevance of a genotoxic effect or exclude it in the case of a negative result; a reasonable test battery was required, technically oriented towards the already harmonized international, hierarchical evaluation for toxicological assessment of chemicals. Therefore, an important aim of this project was to define a strategy for the genotoxicological assessment of anthropogenic trace substances. The basic test battery for genotoxicity of micropollutants in drinking water needs to fulfill several requirements. Although quick test results are needed for the determination of HRIV, a high degree of transferability to human genotoxicity should be ensured. Therefore, an in vitro genotoxicity test battery consisting of the Ames fluctuation test with two tester strains (ISO 11350), the umu test and the micronucleus test, or from the Ames test with five tester strains (OECD 471) and the micronucleus test is proposed. On the basis of selected test substances, it could be shown that the test battery leads to positive, indifferent, and negative results. Given indifferent results, the health authority and the water supplier must assume that it is a genotoxic substance. Genetically modified tester strains are being sensitive to different chemical classes by expression of selected mammalian key enzymes for example nitroreductase, acetyltransferase, and glutathione-S-transferase. These strains may provide valuable additional information and may give a first indication of the mechanism of action. To check this hypothesis, various additional strains expressing specific human-relevant enzymes were investigated. It could be shown that the additional use of genetically modified tester strains can enhance the detectable substance spectrum with the bacterial genotoxicological standard procedures or increase the sensitivity. The additional use provides orienting information at this level as a lot of data can be obtained quite quickly and with little effort. These indications of the mechanism of action should be however verified with a test system that uses mammalian cells, better human cells, to check their actual relevance. The selection of appropriate additional tester strains has to be defined from case to case depending on the molecular structure and also still requires some major expertise.

Occurrence and fate of nitrification and urease inhibitors in the aquatic environment.

Nitrification and urease inhibitors (NUIs) decelerate the bacterial oxidation of nitrogen species by suppressing the activity of soil microorganisms. Thus, nitrogen losses can be limited and the efficiency of nitrogen fertilizers can be increased. After application NUI transfers to surface water may occur through leaching or surface run-off. In order to assess the occurrence of nitrification and urease inhibitors in the aquatic environment a multi-analyte high-performance liquid chromatography-mass spectrometry method was developed. 1H-1,2,4-Triazole and dicyandiamide (DCD) were detected for the first time in German surface waters. Only at a few sites 1H-1,2,4-triazole has been episodically detected with concentrations up to the µg L(-1)-range. DCD was ubiquitously present in German surface waters. An industrial site was identified as the point source of DCD being responsible for exceptionally high DCD concentrations of up to 7.2 mg L(-1) in close proximity to the point of discharge. Both compounds were also detected in at least one wastewater treatment plant effluent, but their concentrations in surface waters did not correlate with those of typical markers for domestic wastewater. Other NUIs were not detected in any of the samples. Laboratory-scale batch tests proved that 1H-1,2,4-triazole and DCD are not readily biodegradable, are not prone to hydrolysis and do not tend to adsorb onto soil particles. Ozonation and activated carbon filtration proved to be ineffective for their removal.

Characterization of a new endogenous vitamin A metabolite.

Here, we describe the discovery of a new major endogenous vitamin A metabolite with particularly high hepatic concentrations. This metabolite was isolated from mouse livers and was characterized as 9-cis-4-oxo-13,14-dihydro-retinoic acid (RA) based on mass spectral, ultraviolet, and nuclear magnetic resonance analyses. It was also detected in one human liver. To gain further insight into endogenous retinoid metabolism, mice were fed over a period of 14 days ad libitum with diets enriched with different amounts of retinyl palmitate [15,000, 45,000 or 150,000 international units (IU)/kg diet]. Higher retinyl palmitate amounts in the diet resulted surprisingly in a dose-dependent decrease in all-trans-RA levels in serum, kidney, and brain, whereas levels of 9-cis-4-oxo-13,14-dihydro-RA, retinol, and retinyl esters were dose-dependently elevated in serum, kidney, and liver. 13-cis-RA levels could be detected in serum, liver, and kidney, but were unaffected by the dietary vitamin A status. 9-cis-RA levels were below the detection limit of 0.2 ng/ml serum or 0.4 ng/g tissue. This study indicates that the oxidation at C4 of the cyclohexenyl ring, isomerization of the C9/C10 double bond, and reduction of the C13/C14 double bond are major endogenous metabolic pathways of vitamin A.

Retinoid status and responsiveness to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking retinoid binding protein or retinoid receptor forms.

We have investigated the role of Vitamin A (retinoid) proteins in hepatic retinoid processing under normal conditions and during chemical stress induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a chemical known to interfere with retinoid turnover and metabolism. Three separate studies were performed in wildtype control mice and transgenic mice that lack one or more isoforms of retinoic acid receptors (RAR), retinoid X receptors (RXR), or intracellular retinoid-binding proteins (CRABP I, CRABP II, CRBP I). Body and organ weight development was monitored from 2 weeks of age to adult, and hepatic levels of retinyl esters, retinol, and retinoic acid were investigated. In addition, hepatic concentrations of 9-cis-4-oxo-13,14-dihydro-retinoic acid, a recently discovered retinoid metabolite that has proven sensitive to both TCDD exposure and Vitamin A status, were also determined. Mice absent in the three proteins CRBP I, CRABP I, and CRABP II (CI/CAI/CAII-/-) displayed significantly lower hepatic retinyl ester, retinol, and all-trans-retinoic acid levels compared to wildtype mice, whereas the liver concentrations of 9-cis-4-oxo-13,14-dihydro-retinoic acid was considerably higher. After treatment with TCDD, hepatic total retinoids were almost entirely depleted in the CI/CAI/CAII-/- mice, whereas wildtype mice and mice lacking CRABP I, and CRABP II (CAI/CAII-/-) retained approximately 60-70% of their Vitamin A content compared to controls at 28 days. RAR and RXR knockout mice responded similarly to wildtype mice with respect to TCDD-induced retinoid disruption, with the exception of RXRbeta-/- mice which showed no decrease in hepatic Vitamin A concentration, suggesting that the role of RXRbeta in TCDD-induced retinoid disruption should be further investigated. Overall, the abnormal retinoid profile in the triple knockout mice (CI/CAI/CAII-/-), but not double knockout (CAI/CAII-/-) mice, suggests that a loss of CRBP I may account for the difference in retinoid profile in CI/CAI/CAII-/- mice, and is likely to result in an increased susceptibility to hepatic retinoid depletion following dioxin exposure.

Selection of organic process and source indicator substances for the anthropogenically influenced water cycle.

An increasing number of organic micropollutants (OMP) is detected in anthropogenically influenced water cycles. Source control and effective natural and technical barriers are essential to maintain a high quality of drinking water resources under these circumstances. Based on the literature and our own research this study proposes a limited number of OMP that can serve as indicator substances for the major sources of OMP, such as wastewater treatment plants, agriculture and surface runoff. Furthermore functional indicators are proposed that allow assessment of the proper function of natural and technical barriers in the aquatic environment, namely conventional municipal wastewater treatment, advanced treatment (ozonation, activated carbon), bank filtration and soil aquifer treatment as well as self-purification in surface water. These indicator substances include the artificial sweetener acesulfame, the anti-inflammatory drug ibuprofen, the anticonvulsant carbamazepine, the corrosion inhibitor benzotriazole and the herbicide mecoprop among others. The chemical indicator substances are intended to support comparisons between watersheds and technical and natural processes independent of specific water cycles and to reduce efforts and costs of chemical analyses without losing essential information.

PAV-1, a new rat hepatic stellate cell line converts retinol into retinoic acid, a process altered by ethanol.

During liver fibrogenesis or long term culture, hepatic stellate cells (HSCs) evolved from "quiescent" to activated phenotype called "myofibroblast-like", a transition prevented by retinoic acid (RA). Little is known about RA generation by HSCs. Our study aimed to check the ability of these cells to produce RA from retinol (Rol) and the alterations of this metabolic step by ethanol. To study this metabolic pathway, primary cultures of HSCs represent the most physiological model but technically suffer several drawbacks. To circumvent these problems, an immortalized rat HSC line (named PAV-1) has been established. We validated PAV-1 cell line as a convenient model to study retinoids metabolism by HSCs. Then, we showed that PAV-1 cells express Rol-binding proteins (RBPs), enzymes and nuclear receptors involved in RA signaling pathway. We also demonstrated in situ generation of functional all-trans-RA (ATRA), using transient transfections with a RA-sensitive reporter gene, in situ modulation of tissue transglutaminase (tTG) activity and HPLC experiments. This production was Rol dose-dependent; 4-methylpyrazole, citral, and ethanol-inhibited which argues in favor of an enzymatic process.In conclusion, we first demonstrate in situ RA generation from Rol in a newly immortalized rat HSC line, named PAV-1. Inhibition of RA production by ethanol in PAV-1 and recent data, suggesting fundamental role of RA to prevent fibrosis development in the liver, allow us to hypothesize that Rol metabolism could be a primary target for ethanol during development of hepatic fibrosis.

2,3,7,8-tetrachlorodibenzo-p-dioxin induces lecithin: retinol acyltransferase transcription in the rat kidney.

Vitamin A (retinoids) has an essential role in development and throughout life of humans and animals. Consequently, effects of the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on retinoid metabolism may be contributory to its toxicity. This study was performed to clarify the mechanism behind dioxin-induced retinyl ester formation in the rat kidney. In addition we investigated the possible role of CYP1A1 in dioxin-induced all-trans-retinoic acid (atRA) formation. Male Sprague-Dawley rats were exposed to a single oral dose of TCDD in a combined dose-response and time-course study, with doses ranging from 0.1 to 100 microg/kg bw and time points from 1 to 28 days. Levels of atRA and the expression of two potentially retinoic acid (RA)-controlled proteins critically involved in retinoid storage regulation, lecithin: retinol acyltransferase (LRAT) and cellular retinol binding protein I (CRBP I), were analyzed in liver and kidney. The expression and activity of cytochrome P4501A1 (assayed as ethoxyresorufin-O-deethylase activity) was assessed to gain insight into its potential role in RA synthesis. There was a significant increase in LRAT mRNA expression in the kidney, whereas no such increase could be observed in the liver, despite significantly increased atRA levels in both tissues. This suggests a tissue-specific regulation of LRAT by TCDD that may be dependent on other factors than atRA. Neither CRBP I mRNA nor protein levels were altered by TCDD. The time-course relationship between CYP1A1 activity and atRA levels in liver and kidney does not exclude a role of CYP1A1 in TCDD-induced RA synthesis. The observed altered regulation of the retinoid-metabolizing enzyme LRAT, together with the low doses and short time required by TCDD to change tissue RA levels, suggest that enzymes involved in retinoid metabolism are specific and/or direct targets of TCDD.

Occurrence of aminopolycarboxylates in the aquatic environment of Germany.

Aminopolycarboxylic acids, such as ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), 1,3-propylenediaminetetraacetic acid (1,3-PDTA), beta-alaninediacetic acid (beta-ADA), and methylglycinediacetic acid (MGDA), are used in large quantities in a broad range of industrial applications and domestic products in order to solubilize or inactivate various metal ions by complex formation. Due to the wide field of their application, their high polarity and partly low degradability, these substances reach the aquatic environment at considerable concentrations (in the microg/L-range) and have also been detected in drinking water. This review evaluates and summarizes the results of long-term research projects, monitoring programs, and published papers concerning the pollution of the aquatic environment by aminopolycarboxylates in Germany. Concentrations and loads of aminopolycarboxylates are presented for various types of water including industrial and domestic waste waters, surface waters (rivers and lakes), raw waters, and drinking waters.

Perfluorinated compounds in the vicinity of a fire training area--human biomonitoring among 10 persons drinking water from contaminated private wells in Cologne, Germany.

In Cologne, Germany, increased concentrations of perfluorinated compounds (PFC) have been observed in two private wells used for drinking water purposes. Both wells are located in the vicinity of a fire training area. Use of well water as a source of drinking water was prohibited by the Public Health Department of the City of Cologne. A human biomonitoring (HBM) survey was performed among all persons, who consumed water from these private wells (N=10). PFC concentrations in water of the private wells and in blood samples were analysed by tandem mass spectrometry with electrospray ionization (LC-ESI-MS/MS). Repeated water analyses (seven measurements between December 2009 and November 2010) indicated a decrease of PFOS from 8.35 to 1.60 µg/l, (PFHxS: 2.36-0.15 µg/l; PFOA: 0.16-0.03 µg/l) in one private well. Although situated close together, PFC-concentrations in the other private well were significantly lower. PFOS-concentrations in blood samples of private well water consumers ranged from 4.8 to 295 µg/l (PFHxS: 12.1-205 µg/l; PFOA: 4.0-18 µg/l). Although no data on the formulation of the firefighting foams applied on the fire training area is available, firefighting foams are supposed to be the most likely source of contamination. These findings give reason to track systematically the application of PFC-containing firefighting foams in order to identify contaminations of surface, ground and drinking waters.

N,N-dimethylsulfamide as precursor for N-nitrosodimethylamine (NDMA) formation upon ozonation and its fate during drinking water treatment.

Application and microbial degradation of the fungicide tolylfluanide gives rise to a new decomposition product named N,N-dimethylsulfamide (DMS). In Germany, DMS was found in groundwaters and surface waters with typical concentrations in the range of 100-1000 ng/L and 50-90 ng/L, respectively. Laboratory-scale and field investigations concerning its fate during drinking water treatment showed that DMS cannot be removed via riverbank filtration, activated carbon filtration, flocculation, and oxidation or disinfection procedures based on hydrogen peroxide, potassium permanganate, chlorine dioxide, or UV irradiation. Even nanofiltration does not provide a sufficient removal efficiency. During ozonation about 30-50% of DMS are converted to the carcinogenic N-nitrosodimethylamine (NDMA). The NDMA being formed is biodegradable and can at least partially be removed by subsequent biologically active drinking water treatment steps including sand or activated carbon filtration. Disinfection with hypochlorous acid converts DMS to so far unknown degradation products but not to NDMA or 1,1-dimethylhydrazine (UDMH).

Contribution of cellular retinol-binding protein type 1 to retinol metabolism during mouse development.

Within cells, retinol (ROL) is bound to cytoplasmic proteins (cellular retinol-binding proteins [CRBPs]), whose proposed function is to protect it from unspecific enzymes through channeling to retinoid-metabolizing pathways. We show that, during development, ROL and retinyl ester levels are decreased in CRBP type 1 (CRBP1) -deficient embryos and fetuses by 50% and 80%, respectively. The steady state level of retinoic acid (RA) is also decreased but to a lesser extent. However, CRBP1-null fetuses do not exhibit the abnormalities characteristic of a vitamin A-deficiency syndrome. Neither CRBP1 deficiency alters the expression patterns of RA-responding genes during development, nor does CRBP1 availability modify the expression of an RA-dependent gene in primary embryonic fibroblasts treated with ROL. Therefore, CRBP1 is required in prenatal life to maintain normal amounts of ROL and to ensure its efficient storage but seems of secondary importance for RA synthesis, at least under conditions of maternal vitamin A sufficiency.

Impact of aminopolycarboxylates on aquatic organisms and eutrophication: overview of available data.

Aminopolycarboxylic acids, which include ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), 1,3-propylenediaminetetraacetic acid (1,3-PDTA), beta-alaninediacetic acid (beta-ADA), and methylglycinediacetic acid (MGDA), constitute a class of complexing agents that occur in a wide range of domestic products and that are used intensively as metal sequestrants in several industrial applications. Because they are highly polar and partially nondegradable, aminopolycarboxylates are released into the aquatic environment in significant quantities, mainly via wastewater. The historical and current use of aminopolycarboxylates and their ubiquitous presence in surface waters prompted many studies about their possibly detrimental impact on aquatic organisms. This review summarizes the available data and information on the eutrophication potential and toxicity of aminopolycarboxylates to a multitude of aquatic organisms including vertebrates, invertebrates, algae, bacteria, and protozoa. This article also addresses how the ecotoxic effects of aminopolycarboxylates are dependent on their speciation, that is, on their presence in a free or a metal-complexed form.

Chromatographic analysis of endogenous retinoids in tissues and serum.

We present a reliable, highly sensitive, and versatile method for the simultaneous determination of endogenous polar (acidic) and apolar (retinol, retinal, and retinyl esters) retinoids in various biological matrices. Following a single liquid extraction of retinoids from tissues or plasma with isopropanol, polar retinoids are separated from apolar retinoids and neutral lipids via automated solid-phase extraction using an aminopropyl phase. After vacuum concentration to dryness and reconstitution of the residue in appropriate solvents, the obtained fractions are injected onto two different high-performance liquid chromatography (HPLC)-systems. Polar retinoids are analyzed on a RP18 column (2.1mm ID) using a buffered gradient composed of methanol and water and on-column-focusing large-volume injection. Apolar retinoids are separated on a normal-bore RP18 column using a nonaqueous gradient composed of acetonitrile, chloroform, and methanol. Both HPLC systems are coupled with UV detection, and retinoids are quantitated against appropriate internal standards. The method was validated with regard to recovery, precision, robustness, selectivity, and analyte stability. Using 400 microl serum or 200mg tissue, the limits of detection for all-trans-retinoic acid were 0.15ng/ml or 0.3ng/g, respectively. The corresponding values for retinol were 1.2ng/ml or 2.4ng/g, respectively. This method was successfully applied to mouse, rat, and human tissue and serum samples.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the endogenous metabolism of all-trans-retinoic acid in the rat.

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is known to influence vitamin A homeostasis. In order to investigate the mechanism behind this retinoid disruption, male Sprague-Dawley rats were exposed to TCDD at doses ranging from 0.1 to 100 micro g/kg body weight, and were killed 3 days after exposure. Additional groups of rats were killed 1 and 28 days after a single oral dose of 10 micro g TCDD/kg body weight. Serum, kidney, and liver were investigated for retinoid levels, as well as gene expression and enzyme activities relevant for retinoid metabolism. Besides the well known effects of TCDD on apolar retinoids, i.e. decreased hepatic and increased renal retinyl ester (RE) levels, we have found dose-dependent elevation of all- trans-retinoic acid (all- trans-RA) levels in all investigated tissues. In the liver, 9- cis-4-oxo-13,14-dihydro-RA was drastically decreased by TCDD in a dose-dependent manner. In serum, cis-isomers of all- trans-RA, including 9,13-di- cis-RA, were significantly reduced already at the lowest dose level. Protein and mRNA levels of cellular retinol binding protein I (CRBP-I) in liver or kidneys were not significantly altered by TCDD exposure at doses at which retinoid levels were affected, making CRBP-I an unlikely candidate to account for the alterations in retinoid metabolism caused by TCDD. The expression and activities of relevant cytochrome P450 (CYP) enzymes with potential roles in all- trans-RA synthesis and/or degradation (CYP1A1, 1A2, and 2B1/2) were also monitored. A possible role of CYP1A1 in TCDD-induced all- trans-RA synthesis is suggested from the time-course relationship between CYP1A1 activity and all- trans-RA levels in liver and kidney. The significant alteration of the all- trans-RA metabolism has the potential to contribute significantly to the toxicity of TCDD.