Analysis of difference in vivo effects of TCDD between c-src +/+ mice, c-src deficient, -/+ and -/- B6, 129-Src(tm 1 sor) mice and their wild-type littermates.

Toxicities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were tested on two strains of c-src deficient B6, 129-Src(tm 1 sor) mice, c-src -/+ and c-src -/- and their matched wild-type littermates c-src +/+ mice along with another c-src +/+ mice, from the same genetic lineage, B6, 129-Fos(tm 1 Pa) mice. The most conspicuous effect of c-src deficiency on the toxicity of TCDD appears to be the reduced hepatotoxicity. TCDD-treated c-src deficient mice show only modest degrees of hepatomegaly and triglycerides accumulation as compared to treated wild-type mice.

Differential toxicities of TCDD in vivo among normal, c-src knockout, geldanamycin- and quercetin-treated mice.

Although we have previously reported the result of our preliminary study on the reduced toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo in male c-src deficient, -/+ B6, 129-src(tmlSor) mice as compared to those in wild-type mice, there are still two major shortcomings of the above study: (a) the low number of individuals tested, (b) in some of the comparison tests, C57BL/6J mice (i.e. inbred B6 mice) were used as wild-type control mice. Since then we increased our laboratory breeding program and thereby the availability of B6, 129-/+, -/- and true littermate wild-type +/+ individual mice. The results of critical in vivo toxicity comparison tests, involving 6-13 mice per test group, showed that there are considerable variations expressed in toxicity within each group of c-src deficient mice. Nevertheless, when a large enough number of individuals were tested two toxic effects were found to be less expressed in src-deficient mice. They were: (a) excess fatty deposits and (b) the mottled appearance of the liver which were commonly observed in TCDD-treated wild type mice, but not in c-src deficient mice. The former trend was also confirmed by both liver lipid analysis and histological examinations of the affected livers. As for the biochemical parameters, the hepatic nuclear protein binding to C/EBP (CCAAT/enhancer binding protein) response element appears to be uniformly reduced by the action of TCDD in + / + mice, but not in -/+ or -/- mice.

Evidence for organochlorine contamination in tissues of salmonids in Lake Tahoe.

To evaluate the extent of organochlorine pollution in the Sierra Nevada ecosystem, residues of certain organochlorines in lake trout and Kokanee fish from Lake Tahoe, an alpine lake located between the Sierra Nevada Mountain Range and the Carson Range of California and Nevada, were analyzed. Multiresidue analysis in fish muscle revealed wet weight concentrations of total polychlorinated biphenyls (PCBs) in the range 18 to 430 ppb and of p,p'-DDE in the range 5 to 85 ppb in the two fish species studied. In one large lake trout sample (6.6 kg), which was studied in more detail as compared with others, residue levels of PCB (267 ppb), toxaphene (154 ppb), a chlordane mixture (78 ppb), and a DDT mixture (154 ppb) were found in muscle. Full spectra of specific PCB congeners and p,p'-DDE were obtained from fish fat tissues and their identities were confirmed using gas chromatography/mass spectrometry (GC-MS). The results of total PCB analysis indicated that residues found in fish consisted mostly of moderately (tri- to tetrachloro-) to highly (penta- to heptachloro-) chlorinated biphenyls. For all fish residues analyzed, the best match to PCB residue profiles was with Aroclor 1260 or 1262.

Sequences of cDNAs encoding calmodulin, and partial structures of calmodulin kinase, and a calcium channel of kdr-resistant and -susceptible German cockroaches, Blattella germanica.

Complementary DNA sequences of genes encoding calmodulin, partial structures of calmodulin-dependent protein kinase II (CaM-kinase II) and an L-type-like calcium channel al subunit (IVS5-IVS6-EF hand region) were identified and compared between susceptible and kdr strains of German cockroach, Blattella germanica. For this purpose, polymerase chain reactions (PCR) were used to obtain their sequences using cDNA from poly(A) + RNA isolated from their heads and thoraces. No mutation differences were found in all three sequences of calcium-regulating proteins between susceptible and strain. Northern blot analysis, however, showed reduced expressions of CaM-kinase II mRNA in two kdr strains. Western blot analysis with an antibody preparation against CaM-kinase II on protein levels confirmed the above strain difference in the titer of this enzyme. In contrast, the levels of calmodulin as well as that of an L-type-like calcium channel gene expression were not different between susceptible and kdr strains.

Use of c-Src and c-Fos knockout mice for the studies on the role of c-Src kinase signaling in the expression of toxicity of TCDD.

Previously, we reported that several of the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were not fully expressed in c-src -/- and -/+ mice (1). In the current study, we studied the basic molecular mechanism of their differential responses. First, we could show that chemical inhibition of c-src kinase could produce practically the same phenomenon of reduced toxicity of TCDD in wild-type mice cotreated with geldanamycin, a specific chemical inhibitor known to suppress c-src kinase. Second, we established that the level of the Ah receptor associated with c-src kinase (2) is indeed low in c-src deficient mice as well as geldanamycin-treated mice. We could show, at the same time, that the effect of c-src deficiency on the toxicity of TCDD is very selective; that is, despite the reduction of many of its toxic signs, the enlargement of liver, induction of cytochrome P450, and other drug-metabolizing enzymes took place normally in those c-src-deficient mice. Apparently, induction of these detoxification enzymes are independent of c-src-mediated pathway. Based on the known signaling pathways of c-src, we tested c-fos-deficient mice and found that some of the c-src-dependant toxic signs of TCDD such as thymic atrophy and decrease in adipose tissue weight were also reduced in c-fos-deficient mice, indicating that these two toxic effects are likely to be mediated through both c-src and c-fos. However, other TCDD responses noticeable in c-fos-deficient mice; downregulation of receptors for epidermal growth factor (EGF), tumor necrosis factor (TNF alpha), and retinoic acid; and up-regulation of the T3 receptor. These findings clearly show that c-fos mediates only a part of c-src signaling pathway in transducing these specific toxic actions of TCDD as mediated by c-fos.

Suppression of C/EBPalpha and induction of C/EBPbeta by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mouse adipose tissue and liver.

We examined the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on two transcription factors, CAAT/enhancer binding protein-alpha (C/EBPalpha) and beta (C/EBPbeta), involved in the coordination of gene expression in adipose and liver. A single dose of TCDD (100 microg/kg) to male C57BL mice resulted in a time- and dose-dependent decrease in the level of C/EBPalpha mRNA in adipose tissue and liver, and a reciprocal increase in C/EBPbeta mRNA. Gel shift analysis using hepatic nuclear extracts from control and TCDD-treated mice and an oligonucleotide containing a C/EBP recognition element revealed a time-dependent change in DNA-protein complexes formed. Bands corresponding to C/EBPalpha, as determined by supershift analysis, diminished in TCDD-treated animals over a 7-day time period, whereas two new bands corresponding to C/EBPbeta, not present in control extracts, were increased significantly in treated samples. TCDD induced C/EBPbeta mRNA in wild-type mouse hepatoma cells, but not in aryl hydrocarbon receptor (AhR) nuclear translocator-deficient hepatoma cells. Induction in wild-type hepatoma cells was antagonized effectively by a molar excess of alpha-naphthoflavone. These results showed that TCDD caused rapid, reciprocal changes in C/EBPalpha and C/EBPbeta mRNAs and DNA binding in the adipose and liver of male C57BL mice and induced C/EBPbeta in hepatoma cells in an AhR-dependent manner. C/EBPs play vital roles in the coordination of energy homeostasis, and their alteration by TCDD may provide insight into the mechanism by which TCDD perturbs energy storage and utilization in vivo.

Development of broad spectrum antibodies to heat shock protein 70s as biomarkers for detection of multiple stress by pollutants and environmental factors.

To test the hypothesis that broad spectrum antibodies may be developed as biomarkers useful in detecting the consequence of combined environmental stresses in a wide variety of tissues and organisms, a stretch of 16 amino acids, TVPAYFNDSQRQATKDA, a well-conserved portion of heat shock 70 proteins, was identified, against which specific antibodies could be designed. This stretch of peptide was synthetically prepared and used as a hapten for antibody preparation by coupling to keyhole lympet hemocyanin, injecting into a rabbit, collecting its blood, and purifying an IgG-rich fraction. The resulting polyclonal antibody was found to react with many heatshock protein (HSP) 70s in every species tested so far, including two species of fish and one amphibian, two arthropod, and one plant species. To relate the reactivity of this antibody preparation to heat shock proteins known to be induced by environmental stress, a Western blot assay method was used to study several organisms under unstressed or stressed conditions. Invariably, heat treatment caused a rise in the titer or HSP70 and/or glucose-regulated proteins. In addition, in some species chemical stresses were also found to be manifested in the form of an increased titer of these proteins.

Altered in vivo toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C-SRC deficient mice.

Administration of a single i.p. dose of 115 microg/kg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to homozygous and heterozygous c-src deficient mice (i.e. c-src -/- and -/+ mice) and their wild-type littermates (c-src +/+ mice) induced differential toxic responses. In c-src +/+ mice, there were clear-cut signs of the toxicity of TCDD, such as the loss of weight in the body, thymus and adipose tissue, whereas in c-src -/+ mice these effects were modest and were not statistically significant. Yet, hepatomegaly, a characteristic effect of TCDD, took place in all three strains of mice. Histological examination of liver samples from control mice and from mice treated with TCDD for 10 days showed that there are qualitative differences in the expression of the effects of TCDD between control and treated mice as well as between c-src -/+ and +/+ mice. In the case of c-src +/+ mice, the predominant lesions were lipid accumulation, glycogen depletion, edema formation and necrosis, as shown by the presence of large areas of ballooning degeneration, and cellular influx of fluid. These changes were demonstrated only marginally in c-src -/+ mice. The predominant effect in -/+ mice was edema formation. At a high dose of TCDD (345 microg/kg), all of the +/+ mice died within 34 days, whereas none of the c-src -/+ mice died. Together these results clearly indicate that some of the toxic effects of TCDD are not fully expressed in c-src deficient mice.

Cloning and sequencing of the para-type sodium channel gene from susceptible and kdr-resistant German cockroaches (Blattella germanica) and house fly (Musca domestica).

Using reverse transcription polymerase chain reactions (RT-PCR), the DNA sequence for the main membrane-spanning region (IS3 through IVS6) of the gene encoding the alpha-subunit of the para sodium channel of the German cockroach, Blattella germanica, has been determined. The overall structure of the open reading frame region of this B. germanica gene is very similar to that of the para gene of Drosophila melanogaster, and that of the partially sequenced para gene of Musca domestica. On the other hand, it is distinctly different from that of the DSC gene (Drosophila sodium channel). As a result of a side-by-side comparison of the para gene sequences of the susceptible CSMA strain and the kdr resistant VT strain of B. germanica, one mutation (TTG to TTC) at the approximate center of the IIS6 membrane-spanning segment was found to result in an amino acid change from L to F. While the functional meaning of this mutation for the operation of the para sodium channel remains to be studied, this region is very highly conserved among all sodium channels identified so far, and is one of the most hydrophobic areas of the entire alpha-subunit. For comparison, we have studied the same region of the para sodium channel of both kdr and susceptible SBO strain of the housefly, Musca domestica. We found the homologous type of mutation, CTT to TTT, resulting in the same amino acid alteration (L to F) at this site. However, in the case of houseflies both kdr and susceptible strains contained both L and F versions of the protein. The ratio of TTT to CTT was significantly higher in the kdr strain of M. domestica than in the three susceptible strains examined.