Modulation of ethanol toxicity by Asian ginseng (Panax ginseng) in Japanese ricefish (Oryzias latipes) embryogenesis.

Alcohol consumption by women during pregnancy often induces fetal alcohol spectrum disorder (FASD) in children who have serious central nervous system (CNS), cardiovascular, and craniofacial defects. Prevention of FASD, other than women abstaining from alcohol drinking during pregnancy, is not known. A limitation of the use of synthetic anti-alcoholic drugs during pregnancy led us to investigate herbal products. In particular, many plants including Asian ginseng (Panax ginseng) have therapeutic potential for the treatment of alcoholism. We used Japanese ricefish (medaka) (Oryzias latipes), an animal model of FASD, for identifying herbal medicines that can attenuate ethanol toxicity. Fertilized eggs in standard laboratory conditions were exposed to ginseng (PG) root extract (0-2 mg/mL) either 0-2 (group A) or 1-3 (group B) day post fertilization (dpf) followed by maintenance in a clean hatching solution. The calculated IC50 as determined 10 dpf in A and B groups were 355.3±1.12 and 679.7±1.6 µg/mL, respectively. Simultaneous exposure of embryos in sub-lethal concentrations of PG (50-200 µg/mL) and ethanol (300 mM) for 48 h disrupted vessel circulation and enhanced mortality. However, PG (100 µg/mL) may partially protect trabecular cartilage (TC) deformities in the neurocranium in B group embryos induced by ethanol (300 mM). To understand the mechanism, embryonic ethanol concentration was measured at 2 dpf and adh5, adh8, aldh2, aldh9a, catalase, GST, and GR mRNAs were analyzed at 6 dpf. It was observed that although ethanol is able to reduce adh8 and GST mRNA contents, the simultaneous addition of PG was unable to alter ethanol level as well as mRNA contents in these embryos. Therefore, antagonistic effects of PG on ethanol toxicity are mediated by a mechanism which is different from those regulating ethanol metabolism and oxidative stress.

Developmental expression of alcohol dehydrogenase (ADH3) in zebrafish (Danio rerio).

Alcohol dehydrogenase (ADH) is the primary enzyme responsible for metabolism of ethanol to acetaldehyde. One class of ADH has been described in fish, and has been found to be structurally similar to mammalian class III ADH (glutathione-dependent formaldehyde dehydrogenase) but functionally similar to class I ADH (primarily responsible for ethanol metabolism). We have cloned a cDNA by RT-PCR from zebrafish (Danio rerio) liver representing the zebrafish ADH3 gene product, with a coding region of 1131 nucleotides. The deduced amino acid sequences share 90% identity to ADH3 from the marine fish Sparus aurata, and 82 and 81% identity to the mouse and human sequences, respectively. Using a quantitative competitive RT-PCR assay, ADH3 mRNA was detected at all timepoints analyzed and was lowest between 8 and 24 h postfertilization. Thus, differential ADH3 expression may be at least partly responsible for temporal variations in the sensitivity of zebrafish embryos to developmental alcohol exposure.

2,3,7,8-Tetrachlorodibenzo-p-dioxin increases steady-state estrogen receptor-beta mRNA levels after CYP1A1 and CYP1B1 induction in rat granulosa cells in vitro.

Previous in-vitro investigations of rat granulosa cells (GC) have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits estrogen secretion and FSH-induced aromatase activity. Although TCDD exerted no effect on basal aromatase enzyme activity, TCDD did reduce steady-state aromatase mRNA levels in GC using competitive RT-PCR. TCDD is hypothesized to induce these changes through aromatic hydrocarbon receptor(AHR)-mediated gene transcription and the modulation of the estrogen receptor (ER)-signaling pathway. In this study we show that rat GC express mRNA for AHR and the AHR nuclear translocator (ARNT) as well as biomarkers of TCDD action, CYP1A1 and CYP1B1 mRNA. Basal CYP1A1 and ER-alpha mRNAs were present only in trace amounts. By relative RT-PCR analysis we showed that CYP1A1 and CYP1B1 mRNA were induced significantly by TCDD at 6 h and that induction of CYP1A1 was maintained throughout the experiment. Using competitive RT-PCR, we observed no significant change in the mRNA levels of ARNT between control and TCDD-treated GC. Both AHR and ER-beta mRNA levels increased significantly at 48 h with TCDD compared with controls. Since ER-beta mRNA was not increased significantly until 48 h in culture, we suggest that in rat GC, the observed ER-beta mRNA increase by TCDD might be a result of CYP1A1/CYP1B1 catalyzed estrogen metabolism and aromatase mRNA inhibition via AHR.

Demonstration of 2,3,7,8-tetrachlorodibenzo-p-dioxin attenuation of P450 steroidogenic enzyme mRNAs in rat granulosa cell in vitro by competitive reverse transcriptase-polymerase chain reaction assay.

We investigated the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in prepubertal (PP) and adult (A) rat granulosa cells (GC) in vitro by examining the changes in estrogen secretion, aromatase enzyme activity and mRNAs for steroidogenic enzymes P450scc, 3beta-HSDI, P450arom; and for components of the AHR signaling pathway-CYP1A1, aromatic hydrocarbon receptor (AHR), and the AHR nuclear translocator protein (ARNT). In PP and A rat GC, TCDD (3.1 nM) reduced estrogen secretion at 48 h without altering aromatase enzyme activity. Addition of FSH (50 ng/ml) increased aromatase activity in GC with or without TCDD. FSH-induced aromatase activity was significantly reduced by TCDD (3.1 nM) at 48 h. Semi-quantitative RT-PCR showed a significant increase in CYP1A1 mRNA both at 24 and 48 h with TCAP, while a significant reduction in P450scc and P450arom mRNA was observed with competitive RT-PCR. All steroidogenic enzyme mRNAs were significantly lower in adults than in PP GC. We conclude that in rat GC, TCDD modulates the level of cytochrome P450 enzymes involved in the steroid biosynthetic cascade. This effect may be attributable to AHR interaction with dioxin-responsive elements present in the genes encoding these enzymes.

Vitamin B12-induced alterations in activities of alpha-glycerophosphate dehydrogenase and malic enzyme in brain of singi fish, Heteropneustes fossilis (Bloch).

Different doses of vitamin B12 (0.25, 0.5, 1, 2 and 4 micrograms/g, injected intraperitoneally for three consecutive days) altered the activities of mitochondrial-alpha-glycerophosphate dehydrogenase (alpha-GPD) and NADP-dependent cytosolic malic enzyme (ME) in the brain of singi fish. The alpha-GPD activity increased at doses of 0.5, 1, 2 and 4 micrograms/g vitamin B12. A dose of 0.5 microgram/g vitamin B12 induced less activity than higher doses. ME activity increased with 1, 2 and 4 micrograms/g of vitamin B12/g. The mitochondrial and cytosolic protein content remained unchanged after vitamin B12 administration. Cycloheximide treatment inhibited the vitamin B12-induced increase in alpha-GPD and ME activity. Thus, vitamin B12 is involved in the induction of some enzymes in fish brain.

Minimal endocrine alterations in rodents after consumption of lake trout (Salvelinus namaycush).

Polychlorinated biphenyls (PCBs) and dioxins are known to cause disruptions in circulating hormone concentrations, which may influence fertility and normal fetal development. Structure activity relationships have been determined for individual congeners, but it is unclear what impacts occur due to exposure to complex mixtures of chemicals found in the environment. Most laboratory studies of PCB exposure have used commercial mixtures in high doses, which may not be representative of environmental concentrations of individual congeners, nor accurately represent complex interactions of multiple contaminants. The present study investigated endocrine alterations in rats associated with the consumption of lake trout collected from three specific locations in the Great Lakes. Composite fish samples were analyzed for PCBs, organochlorines, and mercury and ranged from 415 ppb to 1,275 ppb for individual contaminants. Fillet composites were fed to timed-pregnant Long-Evans rats as 30% of their diet. Concentrations of total thyroxine and estrogen were not significantly different in offspring of dosed dams from that of controls. However, aromatase activity was lowered in all dosed groups as compared with controls. This may represent a lowered expression of the CYP 19 gene in exposed rats or may be due to the presence of one or more substances in the contaminants that are capable of altering the affinity of the aromatase enzyme for its normal endogenous substrate. It is also possible that the number of maturing follicles in the lake trout-fed rats may be fewer than controls, which would result in an overall reduction in the enzyme activity. Data regarding the endocrine effects of environmental contaminant mixtures found in fish from the Great Lakes Basin are still controversial. Additionally, information is scarce with respect to the F1 generation of laboratory animals following environmental maternal exposures, therefore, we investigated the reproductive-endocrine alterations in rat offspring associated with the consumption of lake trout (Salvelinus namaycush) collected from three areas in the Great Lakes.

Lack of effect of beta-naphthoflavone on induction of Nramp genes in adult rainbow trout Oncorhynchus mykiss.

Natural resistance-associated macrophage protein (Nramp) genes in rainbow trout, Oncorhynchus mykiss, were identified and characterized. The greatest mRNA level encoding these genes was in the developing ovary of rainbow trout. We evaluated the response of these genes to a certain aromatic hydrocarbon receptor (AHR) agonist. Adult rainbow trout were treated with beta-naphthoflavone (BNF) (50 and 100 mg/kg) for 48 h. Using reverse-transcriptase polymerase chain reaction with ovary and head kidney RNA and specific alpha and beta Nramp primers, a 400 bp Nramp-alpha- and a 400 bp Nramp-beta-specific cDNA were obtained. There were no changes in the alpha and beta Nramp mRNA levels in the ovary following BNF administration. CYP1A1 mRNA was increased in the ovary and kidney, suggesting the presence of AHR in rainbow trout ovary, while the AHR agonist produced no effect on Nramp mRNAs.

Down regulation of CYP 1A1 by glucocorticoids in trout hepatocytes in vitro.

Short-term culture of rainbow trout (Onchorhynchus mykiss) hepatocytes was used to examine the effect of dexamethasone (DEX) on microsomal CYP 1A1 protein content and 7-ethoxyresorufin-O-deethylase (EROD) activity in vitro. Hepatocytes prepared by controlled collagenase digestion and plated at a density of 0.25 x 10(6) cells/cm2 in plastic culture dishes precoated with trout skin extract (7.6 micrograms skin protein/cm2) to facilitate cell attachment were maintained at 16 degrees C. Cells were treated with DEX (10(-9) to 10(-7) M) or vehicle (dimethyl sulfoxide, DMSO) at 24 h. Microsomal CYP 1A1 protein content and EROD activities were measured at 72 h. Both CYP 1A1 protein as measured by Western blots using CYP 1A1 specific anti-sera and EROD activity were significantly lower in DEX (10(-8) to 10(-7) M)-treated hepatocytes compared to untreated (control) or DMSO-treated cells. The effect was dose dependent in that a gradual decrease of CYP 1A1 protein and EROD activities were seen with increasing doses of DEX (10(-8) to 10(-7) M). DEX at 10(-9) M was ineffective. Concomitant addition of 10(-6) M RU486, a type II specific glucocorticoid receptor antagonist, to hepatocytes treated with 10(-7) M DEX abolished the DEX effect. RU486 at 10(-8) M was ineffective. Spironolactone (10(-8) to 10(-6) M), a type I specific glucocorticoid receptor antagonist, did not counteract the DEX effect. RU486 or spironolactone (10(-6) M) alone had no effect on CYP 1A1 under similar conditions. DEX thus down regulates CYP 1A1 in fish cultured hepatocytes and this regulation is mediated through the type II glucocorticoid receptor(s).

Stimulation of acetylcholinesterase activity by triiodothyronine in the brain of Singi fish, Heteropneustes fossilis (Bloch).

Three consecutive days of injections of triiodothyronine (T3)(0.038, 0.075, 0.15 and 1.54 nmoles/g) significantly elevated the acetylcholinesterase (AchE) activity in the brain of Singi fish, Heteropneustes fossilis (Bloch). The higher doses of 0.075, 0.15 and 1.54 nmoles of T3/g induced a greater increase in enzyme activity than 0.038 nmoles/g. A T3 dose of 0.019 nmoles/g was found to be ineffective. The T3 action on AchE activity was blocked by cycloheximide. Thiourea treatment for 30 days decreased the AchE activity below the control level. This reduced level of the enzyme activity was brought back even above the control level by T3 injections. It is, therefore, suggested that thyroid hormone is involved in the sustenance of AchE activity in fish brain.

Alterations of ion-dependent ATPase activities in the brain of Singi fish, Heteropneustes fossilis (Bloch), by triiodothyronine.

The activities of Na+K(+)- and Mg(2+)-ATPases in mitochondrial, microsomal, and cytosolic fractions of Singi fish (Heteropneustes fossilis Bloch) brain were investigated after injections of various doses (0.012, 0.025, 0.05, and 0.10 micrograms/g) of triiodothyronine (T3) for 3 consecutive days. Both ATPases were found in the mitochondrial and microsomal fractions. The cytosolic fraction showed only Mg(2+)-ATPase activity. Mitochondrial Na+K(+)-ATPase activity increased to almost the same level in fish treated with 0.025, 0.05, or 0.10 micrograms of T3/g, while the T3 dose of 0.012 micrograms/g was ineffective in this respect. Microsomal Na+K(+)-ATPase activity increased to about the same level with all of the doses of T3 used. No detectable amount of Na+K(+)-ATPase was found in the brain cytosolic fraction. Mitochondrial Mg(2+)-ATPase activity was enhanced with 0.025, 0.05, and 0.10 micrograms of T3/g. The last dose, however, produced a higher increase in activity than the other two doses. Surprisingly, microsomal and cytosolic Mg(2+)-ATPase activity was not increased by T3 treatment. Although T3 concentrations rose sharply after each T3 injection, the serum T3 level in T3-injected fish was not different from that in the control as observed on the fourth day. The T3-induced rise of Na+K(+)- and Mg(2+)-ATPase activities was inhibited by cycloheximide treatment. Immersion of Singi fishes in thiourea significantly reduced brain Na+K(+)-ATPase activity in microsomal and mitochondrial fractions but decreased Mg(2+)-ATPase activity only in the mitochondrial fraction. Three consecutive daily injections of T3 (0.10 micrograms/g) into the thiourea-treated fishes increased their ATPase activities even beyond the control level.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation, stabilization, and molecular weight estimation of thyroid hormone receptors of tadpole and chick embryo erythrocytes.

Typical procedures for the isolation of triiodothyronine (T3) receptors from mammalian nuclei involve extraction of nuclei with buffers containing divalent cations and 0.40M KCl. However, when applied to tadpole erythrocyte (RBC) nuclei, this method gave low yields of relatively unstable T3 receptors. The use of EDTA (10 mM) and 0.4M KCl in a sucrose-Tris buffer resulted in the extraction of 90% of the specifically bound [125I]-T3 from RBC nuclei. It was also found that 5 mM thiol reagent (DTT, GSH, or beta-mercaptoethanol) was required for maximal stability of the receptor. Fractionation of labeled RBC nuclear extracts on a Sephadex G-100 column yielded only one peak of specific T3 binding activity. The T3 receptor peak eluted at the same position as bovine serum albumin (BSA), with an estimated mol wt of 68 kDa. Specific T3 binding activity was destroyed by protease digestion but not by DNAse or RNAse. Scatchard analysis of the fractions from the receptor peak supported the existence of one class of T3 binding sites, with an estimated Kd (about 7 pM) comparable to the Kd reported for the intact RBC. Using the same methods, T3 receptors from the nuclei of chick embryo RBCs were also isolated, again with a Kd (7 pM) similar to that for the intact RBC. The chick receptor also eluted from the Sephadex G-100 column at the same position as BSA. The estimated mol wt of the T3 receptors from both sources is comparable to those reported for T3 receptors from other sources. The results show that T3 receptors derived from both tadpole and chick RBC nuclei could be isolated in a soluble and stable form with no apparent change in Kd.

Demonstration of putative thyroid hormone receptor in the brain nuclei of Singi fish, Heteropneustes fossilis (Bloch).

A single injection of [125I]triiodothyronine (T3) with or without stable T3 in Singi fish, Heteropneustes fossilis (Bloch), showed that the fish brain has saturable binding sites and that the specific uptake is 60-70% higher than the nonspecific uptake. The distribution kinetics of [125I]T3 in the serum, whole brain, and brain nuclei after a single injection of the labeled hormone showed that the removal of [125I]T3 from the serum was very rapid with the t1/2 of about 3.3 hr and the incorporation of the hormone into the brain and brain nuclei were very slow and achieve a maximal value after 4-6 hr of postinjection. The binding of [125I]T3 to the isolated brain nuclei of Singi fish was further studied in vitro. Binding was linearly increased with the increasing concentration of the DNA (nuclei). The binding achieved equilibrium between 15 and 20 min at 27 degrees and was stable at least for 1 hr. The binding was reversible in the presence of excess unlabeled T3. Scatchard analysis showed only a single class of binding sites. The mean dissociation constant (Kd) is 2.15 +/- 0.45 x 10(-10) M and maximum binding capacity (MBC) is 0.044 +/- 0.024 pmol/mg DNA. The relative binding affinities of thyroid hormone analogs for T3 sites were as follows: TRIAC greater than T3 greater than TETRAC greater than T4 greater than reverse T3 greater than T2. These findings were similar to those for other animals. Therefore, the nuclear binding sites in Singi fish brain, as demonstrated, may be regarded as thyroid hormone receptors.

In vitro demonstration of putative nuclear 3,5,3'-triiodothyronine receptors in isolated liver nuclei of Singi fish, Heteropneustes fossilis (Bloch).

Putative thyroid hormone (TH) receptors have been demonstrated in the isolated liver nuclei of Singi fish, Heteropneustes fossilis (Bloch), and their binding characteristics have been examined. Nuclear T3 saturation analyses were carried out in vitro at 27 degrees C in a sucrose-Tris-HCl buffer (pH 7.5) containing calcium (2 mM), magnesium (3 mM) and 2-mercaptoethanol (5 mM). After incubation the bound and free hormones were separated by centrifugation and the nuclei were treated with Triton X-100 (final concentration 0.25%) to reduce the non-specific binding. The binding was saturable and reached equilibrium by 20 minutes of incubation and was also stable for 2 hours. The binding was reversible and the rate of dissociation was more or less equal to the rate of association. The binding was linearly increased with the increased concentrations of the DNA (nuclei). Scatchard analyses of the equilibrium binding data revealed that only one class of binding sites for T3 did exist in the hepatic nuclei of Singi fish. The affinity of these sites or the mean dissociation constant (Kd = 0.20 +/- 0.07 x 10(-10) M) and the mean maximum binding capacity (MBC = 0.17 +/- 0.04 pmol/mg DNA) were in reasonable agreement with the values reported for other teleost fishes.

Effect of lead, zinc, mercury, and copper with and without estrogen on serum vitellogenin level in Magur fish (Clarias batrachus L.).

Injection(s) of lead, zinc, and mercuric acetate decreased the serum vitellogenin content in Magur fish, while cupric acetate failed to cause any change in the vitellogenin level. Estrogen injections on 7th, 8th and 9th d increased the serum vitellogenin level in normal and copper salt treated fish, but were totally ineffective in altering the reduced vitellogenin content in lead, zinc, and mercury salts treated fish. Vitellogenin level almost restored to normal level at 6 week in lead, zinc, and mercury treated fish, and estrogen injections on 37th, 38th, and 39th d enhanced the serum vitellogenin content in all groups.

Effect of 17 beta-estradiol on different parts of central nervous system of female Singi fish (Heteropneustes fossilis Bloch) in relation to reproductive stages.

Effects of different doses of 17 beta-estradiol (1, 2 and 4 micrograms/g, 3 consecutive days injections) on the protein, RNA and DNA contents of cerebrum (CB), cerebellum (CE), midbrain (MB), medulla oblongata (MO) and spinal cord (SC) of female non-vitellogenic (NV) and vitellogenic (V) Singi fish (Heteropneustes fossilis Bloch) were investigated. The amounts of these macromolecules in all these substructures of the central nervous system were enhanced on the 4th and/or 7th day in NV fish by estradiol depending on the dose. The higher dose(s) caused more marked effect. The dose of 1 microgram/g was ineffective in case of protein and mostly in case of DNA. There was no enhancement of protein content with any dose of estradiol on the 4th day in CE, MB, MO and SC, but in CB 4 micrograms of estradiol/g increased the protein content on this day. However, the increase was marked on the 7th day in all substructures. The enhancement of RNA content was elicited earlier (4th day) even with lower dose of 1 microgram/g in NV fish in most of the substructures, except MO. With exception of this substructure again, the DNA content of any part did not increase with the hormone on the 4th day with 1 microgram of estrogen/g. The changes in protein and nucleic acid contents of the different substructures of central nervous system in V fish with 17 beta-estradiol were mostly opposite to those in NV fish. Depending on the dose and time, protein and RNA contents of these parts decreased with estradiol in V fish. No change in DNA content, however, was found, except MO where this cellular constituent was surprisingly enhanced on the 4th and 7th day with all doses of estradiol used. The spinal cord of V fish did not show any change in RNA and DNA contents with the hormone. Thus a reproductive stage-specificity of estrogen action in fish brain is documented.

A comparative study of copper ion induced lysis of vertebrate red blood cells.

1. Erythrocytes from different vertebrate classes were tested for susceptibility towards copper ion-induced lysis under identical copper ion concentration and per cent cell volumes. 2. The susceptibility towards lysis was found to be correlated with the rate of copper ion entry into the erythrocytes. 3. GSH levels decline in red blood cells at a rate proportional to the rate of copper ion entry. 4. Hemolysis does not seem to be causally related to the level of GSH in the erythrocytes.

Decrease in triiodothyronine binding sites in chick embryo erythrocytes during early development.

Specific thyroid hormone (TH) binding sites have been detected in nuclei of erythrocytes obtained from developing chick embryos. The binding characteristics and relative affinities for TH analogs were those expected of TH receptors. Nuclear triiodothyronine (T3) saturation analysis was carried out in vitro by incubating intact erythrocytes in M199 medium with 3-200 pM [125I]T3 for 1 hr at 37 degrees C or 20-24 hr at 21 degrees C. Nuclei were obtained by centrifugation after lysing the erythrocytes in a stabilizing buffer containing 0.3% saponin, followed by addition of Triton X-100 (final concentration 0.2%) to minimize the nonspecific binding. Scatchard analysis of equilibrium binding data suggested that the nuclei possess a single class of binding sites. The binding is reversible and the rate of dissociation is temperature dependent. T3 and T4 appear to bind to the same sites, but the affinity of T3 was 16 times greater. Among TH analogs tested, Triac had the highest affinity followed by L-T3, D-T3, Tetrac, L-T4, D-T4, T2, and rT3. Serial studies performed on different days of chick embryogenesis demonstrated a rapid and significant decrease of the erythrocyte nuclear T3 receptor. On Day 5, the number of T3 binding sites was maximal at 1600 +/- 100 per nucleus. The number declined steadily until, by Day 20, it had reached about 60 +/- 10 sites/nucleus. RBC from adult and baby chickens had less than 1% as many binding sites as those from Day 5 embryos. There was no significant change in the affinity of the sites (Kd approximately equal to 20 pM at 37 degrees C). The reason for the loss of T3 binding sites during embryogenesis is not known. Since the plasma level of the TH increases during embryogenesis, this may reflect down regulation. Another possibility is that the change in erythrocyte population which occurs during this period involves production of erythrocytes which contain fewer T3 binding sites.

Effect of oestradiol dipropionate and testosterone propionate on protein, RNA and DNA contents and RNase and DNase activity in the liver of the toad (Bufo melanosticus).

A single injection of oestradiol dipropionate increased the HSI and protein, RNA and DNA contents and decreased the RNase and DNase activities of the liver of male and female toads. The minimum effective dose of oestrogen required to induce most of these changes was found to be 1 microgram/g (single injection), but the liver RNA content increased at the dose of 0.5 microgram/g. Oestrogen in a dose of 0.1 microgram/g did not cause any of these changes in male and female toads. Testosterone propionate (0.1, 0.5, 1 or 2 micrograms/g, single injection) was mostly ineffective in these respects, while in male toads higher doses of testosterone (1 and 2 micrograms/g) enhanced the liver RNA content only. The oestrogenic responses occurred earlier in female toads than in males. The liver protein and DNA contents increased from the 3rd day in female and on the 5th day in male toads. The liver RNA reached the higher level from the 2nd day in female and from the 3rd day in male. The RNase and DNase activities were reduced from the 2nd and 3rd day, respectively, in female and on the 5th day in male toads.