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.

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.

High level expression of tissue-nonspecific alkaline phosphatase in the milk of transgenic rabbits.

Alkaline phosphatase is a promising therapeutic agent in the Gram-negative bacterial lipopolysaccharide (LPS) mediated acute and chronic diseases. Contrary to other alkaline phosphatase isozymes, purified tissue-nonspecific alkaline phosphatase (TNAP) is not available in large quantities from tissue sources, which would enable to analyse its efficacy in animal sepsis models. Two transgenic rabbit lines were created by pronuclear microinjection with the whey acidic protein promoter-humanTNAP minigene (WAP-hTNAP). Lactating females of both lines produced biologically active human TNAP. As indicated by fractionation of milk samples the recombinant alkaline phosphatase was associated with the membrane of milk fat globules. Alkaline phosphatase enzymatic activity was two orders of magnitude higher compared to normal human serum levels. The demonstration that this TNAP is physiologically active would provide the clue to use transgenic animals as bioreactor for bulk production of the human tissue-nonspecific alkaline phosphatase in milk. This may be a valuable and possibly viable option with important implication in attenuating LPS mediated inflammatory responses.

Effect of post-mortem changes on peripheral and central whole blood and tissue clozapine and norclozapine concentrations in the domestic pig (Sus scrofa).

Interpretation of the results of psychoactive or other drug measurements in post-mortem blood specimens may not be straightforward, in part because analyte concentrations in blood may change after death. There is also the issue of comparability of plasma (or serum) results to those obtained in whole blood. To investigate these problems with respect to clozapine, this drug (10mg/kg daily) was given orally to two pigs. Blood was collected 3h post-dose on day 7, the animals were sacrificed, and blood taken from central and peripheral veins for up to 48 h after death. Tissue samples were also collected immediately after death and at 48 h. Ante-mortem whole blood clozapine/N-desmethylclozapine (norclozapine) concentrations were 0.86/1.07 and 1.11/1.15 mg/l in pigs 1 and 2, respectively. Blood clozapine and norclozapine concentrations generally increased after death (central vein: clozapine up to 300%, norclozapine up to 460%; peripheral vein: clozapine up to 155%, norclozapine up to 185%). Initial blood and kidney clozapine and norclozapine concentrations were comparable in both animals, but were some two-fold higher in heart, liver and striated muscle in pig 2. In both animals, the heart and striated muscle clozapine and norclozapine concentrations had increased some two- to three-fold at 48 h, whilst the liver and kidney concentrations were essentially unchanged. The reason for the increase in heart and striated muscle concentrations at 48 h is unclear, but could be simple variation in sample site. The plasma:whole blood distribution of clozapine and norclozapine was studied in vitro. In human blood (one volunteer donor, haematocrit 0.50) the plots of plasma versus whole blood concentration were linear for both analytes across the range 0.1-1.5mg/l, although clozapine favoured plasma (plasma:whole blood ratio=1.12), whereas norclozapine favoured whole blood (ratio 0.68). In pig blood, the plots of plasma versus whole blood were non-linear in both cases, although clozapine favoured plasma to a greater extent than norclozapine. This may be due to lower plasma clozapine and norclozapine protein binding capacity in the pig as compared to man.

[The weakest link: medroxyprogesterone acetate in pig feed]. / De Zwakste Schakel: Medroxyprogesteron-acetaat in varkensvoeder.

Medroxyprogesterone acetate (MPA)-contaminated feed arrested the onset of farrowing, and induced post-lactational anoestrus in sows. Sixty percent of the sows developed cystic ovaries after weaning following exposure to pharmaceutical waste of MPA in glucose syrup. This waste ended up in acidified feed of by-products of a sow farm, and proved to be the cause of the disorders. Analysis by thin layer chromatography and Gas Chromatography/Mass Spectrometry of renal fat from 10 slaughter sows demonstrated residues of 2.5-8 ppb of MPA. Within the European Union use of MPA is illegal as growth promoter in production animals, and therefore MPA-exposed farms were placed under official control by the general inspection service. Clinical signs and diagnostic procedures of the initial case are presented and the role of the veterinary practitioner in detecting potential food safety hazards is discussed.

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.

The pharmacokinetics of glycyrrhizic acid evaluated by physiologically based pharmacokinetic modeling.

Glycyrrhizic acid is widely applied as a sweetener in food products and chewing tobacco. In addition, it is of clinical interest for possible treatment of chronic hepatitis C. In some highly exposed subjects, side effects such as hypertension and symptoms associated with electrolyte disturbances have been reported. To analyze the relationship between the pharmacokinetics of glycyrrhizic acid in its toxicity, the kinetics of glycyrrhizic acid and its biologically active metabolite glycyrrhetic acid were evaluated. Glycyrrhizic acid is mainly absorbed after presystemic hydrolysis as glycyrrhetic acid. Because glycyrrhetic acid is a 200-1000 times more potent inhibitor of 11-beta-hydroxysteroid dehydrogenase compared to glycyrrhizic acid, the kinetics of glycyrrhetic acid are relevant in a toxicological perspective. Once absorbed, glycyrrhetic acid is transported, mainly taken up into the liver by capacity-limited carriers, where it is metabolized into glucuronide and sulfate conjugates. These conjugates are transported efficiently into the bile. After outflow of the bile into the duodenum, the conjugates are hydrolyzed to glycyrrhetic acid by commensal bacteria; glycyrrhetic acid is subsequently reabsorbed, causing a pronounced delay in the terminal plasma clearance. Physiologically based pharmacokinetic modeling indicated that, in humans, the transit rate of gastrointestinal contents through the small and large intestines predominantly determines to what extent glycyrrhetic acid conjugates will be reabsorbed. This parameter, which can be estimated noninvasively, may serve as a useful risk estimator for glycyrrhizic-acid-induced adverse effects, because in subjects with prolonged gastrointestinal transit times, glycyrrhetic acid might accumulate after repeated intake.

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.

Distinct immunomodulation by autoimmunogenic xenobiotics in susceptible and resistant mice.

HgCl(2) and diphenylhydantoin (DPH) are prototype chemicals associated with diverse (auto)immune effects in genetically susceptible individuals. Both chemicals activate T cells, and the balance of Th1 versus Th2 activation may influence the clinical outcome of exposure. It is unknown which chemically created neoantigens are responsible for Th activation. We therefore investigated the effect of DPH and HgCl(2) on specific responses to TNP-ovalbumin, in mouse strains with varying sensitivity for the adverse effects. HgCl(2) was found to enhance Th2-driven antibody responses in susceptible B10.s, but protective type 1 responses in resistant B10.d2 mice. This was chemical-specific, as DPH enhanced type 2 responses in both strains. DBA/2 mice were relatively unresponsive to HgCl(2), whereas DPH stimulated type 1 responses in these mice. Interestingly, prior exposure to HgCl(2), but not DPH, facilitated IC deposition in B10.s mice only. Thus, we demonstrate that, depending on MHC-II and background genes, HgCl(2) and DPH preferentially adjuvate type 1 or type 2 responses. In case of HgCl(2), the type of response corresponds with susceptibility to antibody-mediated autoimmunity induced by this chemical. In addition, we demonstrate that, within one strain, different autoimmunogenic chemicals can enhance distinct responses to the same antigen.

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.

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

Newborns are susceptible to hemorrhages (hemorrhagic disease of the newborn or HDN) due to vitamin K deficiency. Induction of cytochrome P450 in the fetal liver by maternal anticonvulsant therapy such as phenobarbital or phenytoin is considered to be a major cause. An observed increase in late hemorrhagic disease (LHD) in breast fed neonates gave rise to the hypothesis that PCBs and dioxins, P450-inducing contaminants present in human milk, might effect vitamin K-dependent blood coagulation. This hypothesis was studied in rats. Administration of a single oral dose of 0.003, 0.03, 0.3, 3 or 30 nmol 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) per kg bodyweight or 0.75, 4, 20, 100 or 500 micromol 2,2',4,4',5,5'-hexachlorobiphenyl/kg bw (HxCB) to female and male rats resulted in dose-related reductions of the vitamin K-dependent coagulation factor VII. The highest factor VII reduction in female rats was 44%, observed after TCDD exposure. The Lowest Observed Adverse Effect Level (LOAEL) of TCDD on female factor VII levels was 0.3 nmol/kg bw (96 ng/kg). There was a significant inverse correlation between Factor VII levels and induction of hepatic ethoxyresorufin O-deethylating (EROD) activity, reflecting CYP1A1, and total P450 content. HxCB had no effect on female coagulation factors. In contrast, in male rats only exposure to HxCB, which induces mainly CYP2B1 and 2B2, decreased both coagulation factors dramatically up to 88%. The LOAEL of HxCB on factor VII in male rats was 100 micromol/kg bw (36 mg/kg). In general, effects on coagulation factors in male rats exceeded those in females. In addition, sex-dependent differences of TCDD and HxCB were observed on the hepatic vitamin K cycle enzyme activities in female and male rats. Vitamin K-dependent (gamma-glutamyl carboxylase activity was mainly induced in female rats; 2.3-fold in the highest dose group of TCDD. In male rats only vitamin K 2,3-epoxide reductase (KO-reductase) activity was induced 1.7-fold by the highest dose of HxCB. KO-reductase activity in female rats was also increased by TCDD, however, less pronounced than the carboxylase activity. Concluding, the hepatic vitamin K cycle still functions and is not blocked by TCDD or HxCB, thus explaining the observed reduction in factor VII. Finally, the possible role of P450 in vitamin K deficiency is discussed. Based on these results it is suggested to investigate the possible role of PCBs and dioxin-like compounds in LHD in more detail.

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.

Characterization of CYP1A in hepatocytes of cynomolgus monkeys (Macaca fascicularis) and induction by different substituted polychlorinated biphenyls (PCBs).

Cynomolgus monkeys (Macaca fascicularis) have been used previously as a model to study effects on cytochrome P450 (CYP) regulation. Until now it has not been elucidated which CYP1A proteins are present in this primate species. The aim of this study was to characterize CYP1A in untreated hepatocytes of cynomolgus monkey using two specific CYP1A inhibitors (alpha-naphthoflavone and furafylline). The effect of different substituted polychlorinated biphenyls (PCBs) on CYP1A regulation was also studied in these hepatocytes. Small quantities of CYP1A2 have been identified in untreated hepatocytes. Northern blots showed the presence of a CYP1A mRNA in untreated hepatocytes, when hybridizations where performed with human CYP1A2 cDNA. Inhibitions with furafylline and alpha-naphthoflavone also suggested the presence of CYP1A2 properties. After induction with different PCBs, (probably) CYP1A1 mRNA and enzyme activity were induced in cynomolgus monkey hepatocytes. As expected, 2,3',4,4',5-PeCB (PCB no. 118), a mono-ortho substituted congener, was a potent CYP1A inducer but 2,2',3,4,4',5',5'-HpCB (PCB no. 180), a di-ortho and 2,2',3,4',5,5',6-HpCB (PCB no. 187), a tri-ortho substituted PCB, could induce CYP1A mRNA and enzyme activity in cynomolgus monkey hepatocytes as well.

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.

Selective immunomodulation by the autoimmunity-inducing xenobiotics streptozotocin and HgCl2.

Exposure to certain drugs and environmental chemicals can provoke the onset of autoimmune disease in susceptible individuals by releasing (self) epitopes for which tolerance has not been established, while simultaneously providing the necessary adjuvant activity. The resulting response type is influenced by the genotype of exposed individuals and relates to susceptibility to the adverse immune effects of the chemicals. Here, we assessed the modulatory role of the chemical compounds themselves. A single injection of streptozotocin (STZ) increased the number of CD8+ cells, macrophages, apoptotic cells, and IFN-gamma-producing T helper and T cytotoxic cells, whereas the number of CD4+ cells and B cells was reduced in the draining lymph node. Coinjection with the reporter antigen TNP-OVA resulted in primary and secondary production of TNP-specific antibodies that were predominantly of IgG2a and IgG2b isotype, whereas STZ did not enhance priming for delayed-type hypersensitivity (DTH) responses to TNP-OVA. Injection of HgCl2 on the other hand, reduced the number of IFN-gamma-producing cells, induced accumulation of B cells and CD4+ and CD8+ T cells, enhanced IgG1 and IgE production to TNP-OVA, and primed for secondary IgG1 and IgE production as well as for DTH reactions. Together these results indicate that a single injection of STZ stimulates type-1 responses, whereas HgCl2 enhanced mixed type-1 and -2 responses in BALB/c mice. These response types match the (auto)immune effects elicited to unknown (auto)antigens following multiple injections of these chemicals.

Effects of some persistent halogenated environmental contaminants on aromatase (CYP19) activity in the human choriocarcinoma cell line JEG-3.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5-pentachlorobiphenyl (PCB126), a technical PCB mixture (Aroclor 1016), and a technical toxaphene mixture (Camphechlor) on aromatase (CYP19) activity were investigated in human choriocarcinoma JEG-3 cells. After 18 h incubation with TCDD, PCB126, Aroclor 1016 or toxaphene, ethoxyresorufin-O-deethylase (EROD), and aromatase activity were determined. To exclude serum effects, incubations were carried out with or without fetal calf serum in the medium. EROD activity was induced by both TCDD and PCB126 in the presence or absence of serum, which indicates that JEG-3 cells are responsive toward dioxin-like chemicals. Neither Aroclor 1016 nor toxaphene affected EROD activity in these cells. Calculated EC50 values for induction of EROD activity were 0.71 and 0.40 nM for TCDD, and 48 and 20 nM for PCB126 in presence or absence of serum, respectively. Incubation with TCDD or PCB126 with or without serum caused a concentration-dependent decrease in the aromatase activity of up to 4.9-fold. Calculated EC50 values for this effect were 52 pM and 13 nM for TCDD, and 75 and 48 nM for PCB126 in the presence and absence of serum, respectively. Aroclor 1016 and toxaphene had no effect on aromatase activity at concentrations up to 1.0 microM for Aroclor 1016 or 3.0 microM for toxaphene. These results show that aromatase activity can be decreased in a concentration dependent way within the same range where EROD activity is increased. In view of these results, possible effects of dioxin-like compounds on estrogen producing and androgen target cells should be studied in more detail.

The use of reporter antigens in the popliteal lymph node assay to assess immunomodulation by chemicals.

Various drugs and other chemicals can induce T-cell-dependent B-cell activation which may lead to allergic or autoimmune-like diseases. Because the nature of the relevant (neo-) antigens is generally not known and probably depends on the chemical, we have explored the potential use of reporter antigens to determine T-cell-dependent B-cell activation by chemicals. TNP-Ficoll and TNP-OVA were used for this purpose because they are recognized by the same TNP-specific B cells, but these cells require distinct costimulation for specific antibody production. It was found that HgCl2, phenytoin, nitrofurantoin, and D-penicillamine stimulated IgG1 production to both antigens, incomplete Freund's adjuvant, silica, and dimethylsulfoxide to TNP-OVA only, and LPS and hydroxyl-amino procainamide to TNP-Ficoll alone. The diabetogene streptozotocin did not enhance IgG1 production, but may enhance a cellular response instead. Tolerogens and a T-cell antigen without intrinsic adjuvant activity did not influence the responses. The IgG1 production to TNP-Ficoll was local and transient, and did not always require T cells. In contrast, responses to TNP-OVA could be measured in serum, led to specific memory, and were strictly T-cell dependent. These results demonstrate that specific antibody production to reporter antigens indicates immunostimulatory effects of chemicals more sensitive than PLN cell count and provides important mechanistic information. Moreover, with TNP-OVA as reporter antigen the kinetics and regulation of chemically enhanced immune responses can be studied without the need to know the relevant neo-antigens for each individual compound.

Ethoxyresorufin-O-deethylase (EROD) inducing potencies of planar chlorinated aromatic hydrocarbons in primary cultures of hepatocytes from different developmental stages of the chicken.

In vitro induction of ethoxyresorufin O-deethylase (EROD) activity in cell cultures is an extensively validated tool for measuring overall potencies of mixtures of halogenated aromatic hydrocarbons (HAHs) in samples from the abiotic or biotic environment. For risk assessment with special attention to effects in wild birds, an assay was developed that makes use of chicken embryo hepatocytes. However, it was questioned whether compound-specific responses are consistent at the various developmental stages. The results of our present study show that there are considerable differences between early and late embryonal and post-hatching stages. The induction of EROD was measured in primary chicken hepatocyte cultures. The cells were isolated at day 14 and day 19 of embryonal development and at day 1 post hatching. Hepatocytes were exposed in vitro to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4',5-pentachlorobiphenyl (PCB 126, IUPAC nomenclature) and 2,3',4,4',5-pentachlorobiphenyl (PCB 118). The respective compounds were chosen as representatives for dioxins, furans, non-ortho PCBs, and mono-ortho PCBs. These groups of chemicals have been identified as environmental contaminants with major dioxin-like effects that are mediated by a common receptor, the arylhydrocarbon (Ah) receptor. At all developmental stages, TCDF was more potent than TCDD. Relative potencies (RP = EC50TCDD/EC50HAH) decreased in the order TCDF < TCDD < PCB 126 < PCB 118. Depending on the developmental stage, TCDF was 1.2 to 3.4 times more potent than TCDD. PCB 126 was equipotent or less potent by a factor of 3 than TCDD. PCB 118 was 100 to 300 times less potent than TCDD. Both the mean effective concentration (EC50) and the maximum EROD activity (Ymax) of all compounds were lower in hepatocyte cultures from 14-day-old embryos than those from 19-day-old embryos or 1-day-old hatchlings. RPs were comparable in 19-day-old embryos and in hatchlings, but significantly different in 14-day-old embryos.

Organotin-induced apoptosis as observed in vitro is not relevant for induction of thymus atrophy at antiproliferative doses.

The organotin compounds di-n-butyltin dichloride (DBTC) and tri-n-butyltin chloride (TBTC) selectively cause thymus atrophy. Previously, DBTC and TBTC were shown to inhibit proliferation of immature thymocytes, but other studies demonstrated that TBTC but not DBTC increased apoptosis in vitro and also in vivo. In this study, we examined whether apoptosis is increased in vitro by DBTC and TBTC at various concentrations and periods of incubation and whether apoptosis is involved in the induction of thymus atrophy at selective antiproliferative doses. In vitro, DBTC or TBTC at a concentration of 3 microM significantly increased DNA fragmentation in freshly isolated rat thymocytes after preincubation for 10 min followed by a 22-hr culture period. After continuous exposure for 22 hr, apoptosis was observed to be optimum at 1 microM TBTC and 0.3 microM DBTC and lower or absent at higher concentrations. Apparently, apoptosis induced by organotins in vitro depends on both duration and concentration of exposure. Selective antiproliferative doses of DBTC nor TBTC increased apoptosis on day 1 or 2 after single oral exposure. In contrast, the corticosteroid dexamethasone caused a depletion of both small and large thymocytes and a marked increase of apoptosis on both days 1 and 2 after dosing. Thus, although apoptosis is involved in the in vitro cytotoxic effects of both organotin compounds, it seems not involved in thymus atrophy at a dose that selectively inhibits immature thymocyte proliferation.