An AMP Kinase-pathway dependent integrated stress response regulates ageing and longevity.

The purpose of this article is to investigate the role of the AMP-kinase pathway (AMPK pathway) in the induction of a concomitant set of health benefits by exercise, numerous drugs, and health ingredients, all of which are adversely affected by ageing. Despite the AMPK pathway being frequently mentioned in relation to both these health effects and ageing, it appears challenging to understand how the activation of a single biochemical pathway by various treatments can produce such a diverse range of concurrent health benefits, involving so many organs. We discovered that the AMPK pathway functions as an integrated stress response system because of the presence of a feedback loop in it. This evolutionary conserved stress response system detects changes in AMP/ATP and NAD/NADH ratios, as well as the presence of potential toxins, and responds by activating a common protective transcriptional response that protects against aging and promotes longevity. The inactivation of the AMPK pathway with age most likely explains why ageing has a negative impact on the above-mentioned set of health benefits. We conclude that the presence of a feedback loop in the AMP-kinase pathway positions this pathway as an AMPK-ISR (AMP Kinase-dependent integrated stress response) system that responds to almost any type of (moderate) environmental stress by inducing various age-related health benefits and longevity.

Development of human cytochrome P450-expressing cell lines: application in mutagenicity testing of ochratoxin A.

With the aim to assess the involvement of distinct forms of cytochrome P450 enzymes in the activation of procarcinogens, we have developed by means of retroviral infection a series of NIH/3T3 cell lines stably expressing human CYP1A1, CYP1A2, CYP2C10, CYP2D6, and CYP2E1 cDNA. The levels of cytochrome P450 enzyme activities were determined using specific substrates. An increase in specific catalytic activity could be observed in all cell lines compared to background activity in vector-infected cells. Furthermore, we developed a test system in which we are able to combine P450-expressing cells with a shuttle vector containing the lacZ' gene, which serves as a reporter gene for mutations. Using this system, we investigated the cytotoxicity and mutagenicity of the mycotoxin ochratoxin A. Natural occurrence of ochratoxin A in food commodities has been linked to an increased incidence of urinary tract tumors in certain geographic regions. Although biotransformation seems to play a crucial role in ochratoxin A toxicity, the possible contribution of metabolites to genotoxicity and carcinogenicity remained unelucidated. We have demonstrated that the mutation frequency of ochratoxin A was increased dependent upon concentration in NIH/3T3 cell lines, stably expressing human CYP1A1, CYP1A2, CYP2C10, and CYP3A4. In contrast, neither in vector-infected NIH/3T3 cells nor in CYP2D6- and CYP2E1-expressing cells was an increase of mutation frequency observed.

Effects of tri-n-butyltin chloride on energy metabolism, macromolecular synthesis, precursor uptake and cyclic AMP production in isolated rat thymocytes.

The inhibitor of oxidative phosphorylation tri-n-butyltin chloride (TBTC) causes membrane damage and disintegration of isolated rat thymocytes at concentrations higher than 1 microM. From a concentration of 0.1 microM, TBTC disturbs energy metabolism as indicated by an increase in methylglucose uptake, glucose consumption and lactate production and by a decrease in cellular ATP levels. Over the same TBTC concentration range, the incorporation of DNA, RNA and protein precursors are markedly reduced. Moreover the production of cyclic AMP upon stimulation of the cells with prostaglandin E1 is effectively inhibited. These effects cannot be explained by an inhibition of nucleoside kinase activity, amino acid uptake or adenylate cyclase activity. The effects of TBTC on macromolecular synthesis and cyclic AMP production are possibly due to a disturbance of the cellular energy state.

Effects of various inhibitors of oxidative phosphorylation on energy metabolism, macromolecular synthesis and cyclic AMP production in isolated rat thymocytes. A regulating role for the cellular energy state in macromolecular synthesis and cyclic AMP production.

Inhibitors of oxidative phosphorylation such as several triorganotin compounds, oligomycin, 2,4-dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone suppress energy metabolism of isolated rat thymocytes as indicated by a reduction of ATP levels, an increase in glucose consumption and by a marked accumulation of lactate. Also these compounds effectively inhibit the incorporation of DNA, RNA and protein precursors into acid-precipitable material of thymocytes. Moreover, the prostaglandin E1-induced elevation of cAMP is markedly reduced by these inhibitors. A correlation is observed between the effects on energy metabolism, macromolecular synthesis and cAMP production, since from a series of trialkyltin chlorides, tri-n-propyltin, tri-n-butyltin and tri-n-hexyltin are very effective inhibitors of these functions, while trimethyltin and tri-n-octyltin affect neither of them; other inhibitors of oxidative phosphorylation, each of them with quite different mechanisms of action, also inhibit macromolecular synthesis and cAMP production. The finding that a rise in intracellular ATP concentrations leads to a reversion of the tri-n-butyltin-induced inhibition of cAMP production and uridine incorporation, indicates a regulating role for the cellular energy state in these aspects of cellular function.

Microdosing of a Carbon-14 Labeled Protein in Healthy Volunteers Accurately Predicts Its Pharmacokinetics at Therapeutic Dosages.

Preclinical development of new biological entities (NBEs), such as human protein therapeutics, requires considerable expenditure of time and costs. Poor prediction of pharmacokinetics in humans further reduces net efficiency. In this study, we show for the first time that pharmacokinetic data of NBEs in humans can be successfully obtained early in the drug development process by the use of microdosing in a small group of healthy subjects combined with ultrasensitive accelerator mass spectrometry (AMS). After only minimal preclinical testing, we performed a first-in-human phase 0/phase 1 trial with a human recombinant therapeutic protein (RESCuing Alkaline Phosphatase, human recombinant placental alkaline phosphatase [hRESCAP]) to assess its safety and kinetics. Pharmacokinetic analysis showed dose linearity from microdose (53 µg) [(14) C]-hRESCAP to therapeutic doses (up to 5.3 mg) of the protein in healthy volunteers. This study demonstrates the value of a microdosing approach in a very small cohort for accelerating the clinical development of NBEs.

Quantification of natural antibody producing B cells in rats by an improved ELISPOT technique using the polyvinylidene difluoride membrane as the solid support.

We describe here a new type of solid support for the ELISPOT assay, the PVDF membrane. In parallel tests, spot yields on this membrane were superior to those obtained with the frequently used nitrocellulose (NC) membrane, coated with the same rat anti-IgM and anti-IgG antibodies, incubated with the same rat spleen cell suspensions, and developed with the same combination of AP-labeled conjugates and substrate. We therefore used the PVDF membrane, coated with anti-rat IgM and IgG antibodies, ssDNA or bromelain-treated mouse erythrocytes (BrMRBC) (exposing phosphatidylcholine (PC) as major autoantigen) to develop ELISPOT assays for the quantification of isotype-specific natural antibody secreting cells (ASC) in rats. We confirmed the isotype specificity of the binding of the anti-rat IgM and anti-rat IgG coating antibodies and conjugates with the secreted rat antibodies in this assay, and, by inhibition of spot formation with soluble antigen, their specificity for ssDNA and BrMRBC. An in-house 18-well culture device for the easy manufacture of PVDF-lined culture wells greatly facilitated coating, blocking, and washing procedures, as compared to the original method in 24 well culture plates. This simple, fast, specific and sensitive ELISPOT assay was used to make an inventory of the numbers of natural splenic ASC in Wistar and Fischer rats.

Synergistic effect of 2,2',4,4',5,5'-hexachlorobiphenyl and 2,3,7,8-tetrachlorodibenzo-p-dioxin on hepatic porphyrin levels in the rat.

We studied the effect of polychlorinated biphenyls (PCBs) on hepatic porphyrin accumulation in female Sprague-Dawley rats by feeding them diets containing 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), 2,3,3',4,4',5-hexachlorobiphenyl (PCB 156), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), or combinations of the single PCB congeners with TCDD for 13 weeks. A dose-dependent increase in hepatic porphyrin accumulation occurred after TCDD, PCB 126, or PCB 156 administration, reaching maximal levels of about twice control values. The lowest dose levels for which a significant increase in hepatic porphyrin accumulation was found were 0.7 microgram TCDD/kg diet, 50 micrograms PCB 126/kg diet, or 6 mg PCB 156/kg diet. These doses are equivalent to 47 ng TCDD/kg/day, 3.2 micrograms PCB 126/kg/day, and 365 micrograms PCB 156/kg/day. Relative potencies for hepatic porphyrin accumulation, using TCDD as a reference, ranged from 0.015 to 0.06 for PCB 126 and from 0.0001 to 0.0003 for PCB 156. CYP1A2 activities significantly correlated with hepatic porphyrin levels, with coefficients of 0.629, 0.483, or 0.808 for TCDD, PCB 126, or PCB 156, respectively. Administration of PCB 153 alone did not result in hepatic porphyrin accumulation. Co-administration of PCB 153 and TCDD revealed a strong synergistic effect on porphyrin accumulation (about 800 times control levels). This synergistic effect was significant in rats fed diets containing any combination of PCB 153 with TCDD. Uroporphyrin III and heptacarboxylic porphyrin were accumulated in porphyrinogenic livers. These results suggest that TCDD induction of CYP1A2 may be involved, leading to oxidation of uroporphyrinogen III to uroporphyrin III, in combination with an increase in delta-aminolevulinic acid synthetase induced by PCB 153. Under porphyrinogenic conditions, an inhibitor of CYP1A2 activity may also be formed. The interactive effects on porphyrin accumulation after co-administration of dioxinlike and non-dioxinlike compounds may have significant implications for the risk assessment of these chemicals.

Physiologically based pharmacokinetic model for tetrachlorobenzyltoluenes in rat: comparison of in vitro and in vivo metabolic rates.

Ugilec 141 is a technical mixture of tetrachlorobenzyltoluenes (TCBTs). It was introduced in the early 1980s as a replacement for polychlorinated biphenyls (PCBs). Based on physicochemical properties and accumulation in the environment, the use of this mixture was prohibited. To gain more insight in the toxicokinetics of these compounds in mammals, rats were exposed to a single iv bolus injection of a mixture of 3 TCBTs. At different time points after dosing, the tissue and blood concentrations of the TCBTs were determined. The adipose tissue is the main storage compartment, followed by skin and muscle. The TCBTs were rapidly eliminated from the liver and the blood, with half lives ranging from 65 to 72 h. Additionally, the tissue concentration data for all 3 TCBTs were analyzed using a physiologically based pharmacokinetic (PB-PK) model. Sensitivity analysis illustrated that the elimination of the TCBTs was not influenced by metabolism only, but also by the blood flow through the liver. Furthermore, the metabolic rates derived from the model were compared to previously reported in vitro metabolic rates. The in vitro values for the TCBTs were only a factor 2 to 3 smaller than the in vivo metabolic rates, indicating the value of in vitro techniques for a priori parameterization of PB-PK models.

Estrogenic potencies of several environmental pollutants, as determined by vitellogenin induction in a carp hepatocyte assay.

Estrogenic potencies of several xenoestrogens were determined in vitro, using cultured hepatocytes from a genetically uniform male carp strain (Cyprinus carpio). Estrogenicity was measured as induction of the yolk protein precursor vitellogenin (Vtg), and compared to Vtg induction by 17beta-estradiol (E2). The order of estrogenic potency was: methoxychlor (MXCL) > o,p-DDT > chlordecone approximately/= bisphenol-A approximately/= 4-t-pentylphenol. Estrogenic potencies of these compounds varied from 1 x 10(-3) to 1 x 10(-4) relative to E2. The synthetic estrogen DES had a relative estrogenic potency of 0.5, whereas dieldrin, beta-endosulfan, o,p-DDE, and toxaphene (technical mixture) did not induce vitellogenesis at concentrations up to 100 microM. Experiments in which cells were simultaneously exposed to E2 and these xenoestrogens showed that the Vtg-inducing activities of E2 and 4-t-pentylphenol or bisphenol-A were (partially) additive, whereas E2 antagonized the estrogenic effects of MXCL and o,p-DDT. The effect of cytochrome P4501A (CYP1A)-induction on the estrogenicity of MXCL was studied by co-exposing cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD (10 pM) caused a greater than 50-fold induction of CYP1A, measured as ethoxyresorufin O-deethylase (EROD) activity, but Vtg induction by MXCL was not significantly affected. This indicates that CYP1A is not involved in the bioactivation of MXCL to more potent estrogenic metabolites in carp. The CARP-HEP (hepatocyte) assay can detect xenoestrogens with a potency > or = 2 x 10(-5) relative to E2. It allows simultaneous testing of more than 10 compounds for both estrogenic and antiestrogenic effects, which makes it a promising tool for the screening of suspected xenoestrogens.

2-Chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity?

There is increasing concern that certain chemicals in the environment can cause endocrine disruption in exposed humans and wildlife. Investigations of potential effects on endocrine function have been limited mainly to interactions with hormone receptors. A need exists for the development of alternate in vitro methods to evaluate chemicals for their potential to disturb various endocrine functions via other mechanisms. Our laboratory is using the human H295R adrenocortical carcinoma cell line to examine chemicals for their potential to interfere with the activity and/or expression of several key cytochrome P450 (CYP) enzymes involved in the biosynthesis of steroid hormones. In this report we demonstrated that the commonly used 2-chloro-s-triazine herbicides atrazine, simazine, and propazine dose-dependently (0-30 microM) induced aromatase (CYP19) activity to an apparent maximum of about 2.5-fold in H295R cells. Basal- and triazine-induced aromatase activity was completely inhibited by the irreversible aromatase inhibitor 4-hydroxyandrostenedione (100 microM). The triazines increased levels of CYP19 messenger ribonucleic acid (mRNA) between 1.5- and 2-fold. The time-response profile of the induction of aromatase activity and CYP19 mRNA by the triazines was similar to that by 8-bromo-cyclic adenosine monophosphate, a known stimulant of the protein kinase-A pathway that mediates the induction of aromatase in these cells. The observed induction of aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, may be an underlying explanation for some of the reported hormonal disrupting and tumor promoting properties of these herbicides in vivo.

The vehicle modulates cellular and humoral responses in contact hypersensitivity to oxazolone.

The development of contact hypersensitivity (CHS) greatly depends on the allergenicity of the inducing agent. However, various cofactors are known to influence the outcome of the response as well. From this perspective, we have compared the effects of five different vehicles: acetone, ethanol, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), and a 4 to 1 mixture of acetone and olive oil (AOO) on the cellular and humoral immune responses to epicutaneously applied oxazolone in female BALB/c mice. A single application of 0.2% oxazolone dissolved in acetone or ethanol induced stronger proliferative responses and higher lymph node cell numbers than the other three vehicles. Moreover, both vehicles led to higher numbers of oxazolone-specific Ab forming cells in the draining lymph nodes of sensitized animals. When the IgG2a/IgG1 ratios were determined to indicate the type of T helper cell involved, the highest values were obtained with AOO and lowest with DMF and DMSO, while acetone and ethanol were in between. Moreover, no correlation was found between oxazolone-specific antibody production and cellular responses, measured as [3H]thymidine incorporation of draining lymph node cells after sensitization and increased ear thickness after challenge. From this study it can be concluded that cellular and humoral responses in CHS to oxazolone are dissimilarly affected by the vehicles used.

A NIH/3T3 cell line stably expressing human cytochrome P450-3A4 used in combination with a lacZ' shuttle vector to study mutagenicity.

An NIH/3T3 cell line, stably expressing human cytochrome P450-3A4 (CYP3A4) cDNA has been developed. This cell line was used in combination with a shuttle vector, containing the bacterial lacZ' gene as reporter gene, to study mutagenicity. Ethylmethanesulphonate and aflatoxin B1 were used as model agents to test this system. The mutation frequency of ethylmethanesulphonate increased concentration dependently and was the same in CYP3A4-expressing cells as in parental NIH/3T3 cells, demonstrating that CYP3A4 activity has no influence on the mutagenicity of ethylmethanesulphonate. The mutation frequency of aflatoxin B1 increased concentration dependently only in the CYP3A4-expressing cells and not in parental nor in vector-transfected cells. This increase in mutation frequency could be completely inhibited by ketoconazole, an inhibitor of cytochrome P450 activity, demonstrating the role of CYP3A4 in the activation of aflatoxin B1. The system described in this paper opens the possibility to study the capacity of single human cytochrome P450s to activate xenobiotics into mutagenic metabolites. Since activation, phase II metabolism, DNA repair and an endpoint for mutations are all present in one cell, this system will be useful in screening as well as in mechanistic studies.

Differential effects of organotin compounds on voltage-gated potassium currents in lymphocytes and neuroblastoma cells.

Effects of organotin compounds were studied on voltage-gated K+ current in whole-cell voltage clamped lymphocytes and in N1E-115 neuroblastoma cells. In human peripheral blood lymphocytes the immunotoxic compounds dibutyltinchloride (DBT, 2.5 microM) and triphenyltinchloride (TPhT, 2.5 microM) decrease the peak amplitude of the K+ current and prolong time to peak. Tributyltinchloride (TBT, 2.5 microM) decreases the K+ current to a greater extent than DBT and TPhT, without affecting the time to peak. The neurotoxic organotin compound trimethyltinchloride (TMT, 2.5 microM) does not affect the voltage-gated K+ current in lymphocytes. Similar effects of DBT were observed in freshly isolated and PHA-activated human lymphocytes and with rat thymocytes. On the other hand, in mouse N1E-115 neuroblastoma cells, none of the organotin compounds altered the voltage-dependent K+ current. In human lymphocytes DBT affects both the peak amplitude and the time to peak of the K+ current in a concentration-dependent manner. At the maximum concentration of 10 microM tested, the peak amplitude of the K+ current was reduced to 22 +/- 4% of the control current. The IC50 and slope factor for block of the peak outward current by DBT amounts to 6.7 +/- 0.4 microM, and 2.7 +/- 0.4, respectively. The delay in K+ current activation does not saturate. At 10 microM DMT increases the time to peak to 332 +/- 12% of the control value. The present results suggest that the effects by DBT originate from two separate interactions with the voltage-gated K+ channel at the extracellular site of the membrane: a direct effect on the closed K+ channel causing a delay in current activation and a membrane-related effect causing inhibition of the K+ current. The differential effects of the organotin compounds may relate to their differential toxicological action.

Detoxification of the estertin stabilizer bis-(beta-carbobutoxyethyl)tin dichloride in rats by hydrolysis of the ester bond.

In a previous comparative toxicity study with alkyltin and estertin stabilizers, it was recognized that estertin compounds displayed in vitro lymphocytotoxic effects comparable to the dialkyltin compounds, but did not induce lymphoid atrophy when administered in vivo to rats as was found for the dialkyltin compounds. This discrepancy between the in vitro and in vivo toxicity of estertin compounds prompted us to study the metabolism of the estertin compound bis-(beta-carbobutoxyethyl)tin dichloride (CBETC) in rats. The hydrolysis product bis-(beta-carboxyethyl)tin dichloride (CETC) was the only metabolite detectable using TLC. After daily intravenous administration of 20 mg CBETC/kg body weight CETC was detected in urine only, whereas no faecal excretion of organotin was found. Intravenous administration of relatively large amounts of 20 mg CETC/kg body weight indicated that this compound is not metabolized but rapidly excreted in urine, probably because of its hydrophilic nature. Daily gavage of 15 mg CBETC/kg body weight resulted in the excretion of appreciable amounts of CETC in urine, but CETC was also found in faeces together with the parent compound. In the gastrointestinal tract CETC would be formed locally probably by acid hydrolysis of CBETC as was shown also in vitro in acidified water. Esterases in the gastrointestinal tract, tissues and blood might also be responsible for the fast hydrolysis of CBETC. As shown in our previous study the hydrolysis product CETC did not cause any lymphocytotoxic effect. Therefore we conclude that in the rat the estertin compound CBETC is effectively detoxified by hydrolysis of the ester bond.

The absorption, tissue distribution and excretion of di-n-octyltin dichloride in rats.

In this study the absorption, tissue distribution and excretion of 14C-labeled di-n-octyltin dichloride ([14C]DOTC) in rats were investigated after oral and intravenous (i.v.) administration. Although after i.v. administration with 1.2 mg [14C]DOTC/kg body weight the tissue radioactivity was about 3-4 times higher than after oral administration with 6.3 mg [14C]DOTC/kg body weight, the relative tissue accumulation was found to be the same after the oral and i.v. dosage. The highest amount of radioactivity was found in liver and kidney, and to a lesser degree in adrenal, pituitary and thyroid glands. The lowest activity was recovered from blood and brain. No selective accumulation was observed in thymus, although it has been reported that thymus atrophy is the most sensitive parameter of DOTC toxicity in rats. For all tissues a time dependent decrease in radioactivity was found, except for kidney. The excretion of radioactivity in feces and urine was determined after a single i.v. or oral dose of 1.2 and 2 mg [14C]DOTC, respectively. After i.v. administration most of the radioactivity was excreted in the feces which was characterized by a biphasic excretion pattern. In orally treated rats more than 80% of the radioactivity was already excreted in the feces during the first day after administration. This indicated that only a small part of the DOTC was absorbed, which was calculated to be approximately 20% of the dose. Similar half-life values of 8.3 and 8.9 days were obtained from the fecal excretion of radioactivity after the i.v. and oral administration, respectively. The urinary excretion of radioactivity appeared to be independent of the body burden, since the daily amount of radioactivity excreted in urine was nearly the same independent of the route of administration as well as the time after administration.

Selective retention of toxic polychlorinated dibenzo-p-dioxins and dibenzofurans in the liver of the rat after intravenous administration of a mixture.

A mixture of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), purified from fly ash from a municipal incinerator, was administered as a single intravenous dose to male rats. Livers were analyzed after 5 h, 1, 2, 4, 7 and 9 days for PCDD and PCDF content. Starting from t = 5 h 2,3,7,8-substituted congeners were the predominant PCDDs and PCDFs retained. 2,3,4,6,7-PnCDF was the only congener without 4 lateral chlorine atoms retained in the liver. For most of the 2,3,7,8-substituted congeners reliable half-lives could not be calculated due to the short experimental period. For only three 2,3,7,8-substituted congeners was a complete elimination from the liver found, within this time period. These congeners were 2,3,7,8-TCDF, 1,2,3,7,8- and 2,3,4,6,7-PnCDF with half-lives of less than 1, 2.1 and 1.6 days, respectively. The other PCDDs and PCDFs, without 4 lateral chlorine atoms, were not detected in the liver from 5 h to 9 days after i.v. administration. Based on the congeneric distribution patterns, it is suggested that besides metabolism, structural specific binding to the Ah-receptor and cytochrome P-450 complex might also be responsible for this selective liver retention.

Absence of interactions on hepatic retention and 7-ethoxyresorufin-O-deethylation activity after co-administration of 1,2,3,7,8-pentachlorodibenzo-p-dioxin and 2,4,5,2',4',5'-hexachlorobiphenyl.

Interactions between 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PnCDD) and 2,4,5,2',4',5'-hexachlorobiphenyl (HxCB) on hepatic retention of PnCDD and on cytochrome P450 related enzyme activities were studied in male C57BL/6J mice. Animals received 8 nmol PnCDD/kg orally, alone or in combination with 1-416 mumol HxCB/kg. Co-administration of HxCB did not alter the hepatic retention of PnCDD or the 7-ethoxyresorufin-O-deethylation (EROD) activity induced by PnCDD as observed after 1 week. A small antagonistic effect on total cytochrome P450 content and 7-pentoxyresorufin-O-depentylation (PROD) activity was observed at a dose of 8 nmol PnCDD/kg and 1 mumol HxCB/kg. Furthermore, a significant induction of PROD activity by PnCDD was found. This was not expected, since PROD activity is considered to be a specific marker for CYP2b related enzyme activity and this type of cytochrome P450 is not induced by polychlorinated dibenzo-p-dioxins such as PnCDD. It is concluded that, under these short-term experimental conditions, no toxicokinetic basis was found to explain the antagonistic effects on hepatic cytochrome P450 related activities observed in the present study or in other studies.

Triorganotin-induced cytotoxicity to rat thymus, bone marrow and red blood cells as determined by several in vitro assays.

To further investigate the immunotoxic effects of tri-n-propyltin chloride (TPTC), tri-n-butyltin chloride (TBTC) and triphenyltin chloride (TPhTC) several cytotoxicity tests with a series of trialkyltin chlorides and TPhTC were carried out, using isolated rat thymocytes as target cells. Thymocytes, cultured in a serum-supplemented medium, were exposed to organotin concentrations ranging from 0.01 to 10 microM for periods up to 30 h. Parameters such as cell count, trypan blue exclusion, chromium release, thymidine incorporation and cyclic AMP production were used to evaluate the cytotoxicity of these compounds. The more lipophilic compounds TPTC, TBTC, tri-n-hexyltin chloride (THTC) and TPhTC appeared most cytotoxic, reducing thymidine incorporation at concentrations as low as 0.05-1 microM. Membrane damage as determined by trypan blue exclusion and chromium release occurred at higher levels (1-10 microM). The water soluble homologue trimethyltin chloride (TMTC) was least effective in all test models. When phosphate-buffered saline supplemented with glucose was used as incubation medium, TBTC appeared more cytotoxic to thymocytes. Using this medium in 5-h incubations the cytotoxicity of TBTC to thymus, bone marrow and red blood cells was compared. Bone marrow cells were slightly less sensitive than thymocytes, while red cells were relatively resistant. In conclusion, of the triorganotin compounds especially the lipophilic homologues are cytotoxic in vitro.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin or 2,2',4,4',5,5'-hexachlorobiphenyl on vitamin K-dependent blood coagulation in female germfree WAG/Rij-rats.

Newborn infants are susceptible to bleeding disorders caused by a vitamin K deficiency, so called 'haemorrhagic disease of the newborn' (HDN). These bleedings often occur in infants after medication of the mother with antiepileptics, such as phenobarbital or phenytoin. It has been suggested that an increase in the late type of HDN in exclusively breast-fed infants might be related to the presence of cytochrome P450-inducing polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) in human milk. In order to study this possible mechanistic relationship 5-week-old, germfree, female WAG/Rij-rats were exposed to a single oral dose of either 1 microgram 2,3,7,8-tetrachlorodibenzo-p-dioxin/kg body weight (TCDD) or 30 mg 2,2',4,4',5,5'-hexachlorobiphenyl/kg body weight (HxCB), representing cytochrome P-450 1A (3-methylcholanthrene type) and 2B (phenobarbital type) inducers. During the experiment blood coagulation time from each rat was measured. Also, hepatic 7-ethoxy-(EROD) and 7-pentoxyresorufin O-dealkylating (PROD) activities and total cytochrome P450 content were measured. Blood coagulation time (Thrombotest) in the HxCB-treated rats was significantly prolonged and positively correlated to PROD activity and total P450 content. No clear effect of TCDD on coagulation time could be observed under these experimental conditions. These results suggest involvement of P450 2B isoenzymes in vitamin K metabolism.

AHTN and HHCB show weak estrogenic--but no uterotrophic activity.

The ubiquitous presence of the polycyclic musks AHTN (6-acetyl-1,1,2,4,4,7-hexamethyltetraline) and HHCB (1,2,4,6,7,8-hexahydro-4,6,6,7,8-hexamethylcyclopenta-gamma-2-b enzopyreen) in surface waters and their identification in human milk fat together with their polycyclic nature, which makes them potential candidates for interference with estrogen receptors, prompted us to assess these compounds for their potential estrogenic effects. We therefore investigated the effects of AHTN and HHCB in ERalpha- and ERbeta-dependent gene transcription assays with Human Embryonal Kidney 293 (HEK293) cells, which have proven to be very suitable to estimate the estrogenic activity of compounds with low binding activity (Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., Van der Saag, P.T., Van der Burg, B., Gustafsson, J.A., 1998. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139, 4252-4264). Both AHTN and HHCB were found to induce a slight but dose-dependent stimulation of transcriptional activity in the transiently ERalpha transfected HEK293 cells. This weak estrogenic response was not observed in the ERbeta transiently transfected cells. However, these cells were less responsive to estradiol than the ERalpha transfected HEK293 cells. Also, no significant increase in transcriptional activity was observed in HEK293 cell lines, permanently expressing the same estrogen-responsive reporter gene construct and either ERalpha or ERbeta. In the classical uterine weight assay performed in juvenile Balb/c mice, no uterotrophic activity of AHTN and HHCB was noted at relatively high dietary exposure levels up to 50 and 300 ppm, respectively, at which levels an increase in liver weight was evident. Also the vitellogenin production by carp hepatocytes, a sensitive marker of estrogenic activity, was not affected by these two fragrance materials (Smeets, J.M.W., Rouhani Rankouhi, T., Nichols, K.M., Komen, H., Kaminsky, N.E., Giesy, J.P., Van den Berg, M., 1999. In vitro vitellogenin production by carp (Cyprimus carpio) hepatocytes as a screening method for determining (anti-) estrogenic activity of xenobiotics. Toxicol. Appl. Pharmacol., 157, 68-76). Therefore it is concluded that these compounds have very weak estrogenic potency, too weak to induce estrogenic effects in wildlife species or humans at the current levels of exposure. These results give further support to the promiscuity of estrogen receptors.