Detoxication of Citrinin with Kojic Acid by the Formation of the Citrinin-Kojic Acid Adduct, and the Enhancement of Kojic Acid Production by Citrinin via Oxidative Stress in Aspergillus parasiticus

Our previous work showed that citrinin (CTN) produced bay Penicillium citrinum inhibited the production of aflatoxin by Aspergillus parasiticus. We also reported that CTN was non-enzymatically converted to a novel CTN-KA adduct with kojic acid (KA) in aqueous condition. We herein observed that unlike CTN, the CTN-KA adduct does not show antimicrobial activity against Escherichia coli or Bacillus subtilis or any cytotoxic effect on HeLa cells, suggesting that CTN was detoxified by KA by the formation of the CTN-KA adduct. To examine the function of KA production by fungi, we isolated A. parasiticus mutants with impaired KA production. When the mutants were incubated in either liquid or agar medium supplemented with CTN, they were more susceptible to CTN than the wild KA-producing strain. The same results were obtained when we used the A. oryzae KA-producing strain RIB40 and KA-non-producing strains. When KA was added to the CTN-containing agar medium, the inhibition of growth by CTN was remarkably mitigated, suggesting that the production of KA protected the fungal growth from CTN's toxicity. We also observed that CTN enhanced the production of KA by A. parasiticus as well as A. oryzae strains. Reverse transcription-PCR showed that CTN enhanced the expression of KA biosynthetic genes (kojA, kojR, and kojT) of A. parasiticus. However, the enhancement of KA production with CTN was repressed by the addition of α-tocopherol or butylated hydroxy anisole, suggesting that KA production is enhanced by oxidative stress via the formation of reactive oxygen species caused by CTN. In contrast, α-tocopherol did not affect inhibition of AF production as well as fungal growth by CTN, suggesting that the regulation of these inhibitions with CTN might be different from that of KA production. We propose a regulation scheme of CTN for each of KA production, AF production, and fungal growth in A. parasiticus.

Deoxynivalenol and Nivalenol Toxicities in Cultured Cells: a Review of Comparative Studies.

The in vitro studies of the toxicities of trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) including cell proliferation, cytokine secretion, and the involvement of heat shock protein 90 (Hsp90) in their toxicities were reviewed. Trichothecene mycotoxins are extremely toxic to leukocytes and leukopenia is one of the leading signs of trichothecene toxicosis, implying that trichothecenes hinder cell proliferation. Both toxins retarded proliferation of all four cell lines tested. NIV was more potent than DON in human promyelocytic leukemia cell line HL60, human lymphoblastic leukemia cell line MOLT-4, and rat aortic myoblast cell line A-10. In contrast, both toxins exhibited almost the same potencies in human hepatoblastoma cell line HepG2. While exposure to 0.3 µg/mL DON greatly induced the secretion of anti-hematopoietic cytokines CCL3 and CCL4, treatment with NIV decreased the secretion of these cytokines in HL60 cells, indicating that the toxicity mechanisms of these mycotoxins differ. Because molecular chaperone Hsp90 occupies a pivotal position in a wide range of pathological processes, the effects of an Hsp90-specific inhibitor radicicol on cytokine secretions were investigated. Radicicol counteracted the effect of DON on cytokine secretion, indicating that Hsp90 plays a crucial role in DON-induced cytokine secretion in HL60 cells. Conversely, the results of co-treatment with NIV and radicicol indicate that radicicol does not mitigate the effect of NIV. Regarding CCL3 and CCL4 secretions, DON and NIV have Hsp90-related and -unrelated mechanisms of toxicities, respectively. Taken together the results suggest that, although these toxins share similar chemical structure, there are differences in their toxic mechanisms.

Rubratoxin-B-induced secretion of chemokine ligands of cysteine-cysteine motif chemokine receptor 5 (CCR5) and its dependence on heat shock protein 90 in HL60 cells.

To elucidate the mechanism underlying rubratoxin B toxicity, the effects of rubratoxin B on the secretion of CCR5 chemokines, CCL3, CCL4, and CCL5, in a human promyelocytic leukemia cell line, HL60, were investigated. In addition, to examine whether the molecular chaperone 90-kDa heat shock protein (Hsp90) contributes to rubratoxin B toxicity, the effects of Hsp90-specific inhibitors, radicicol and geldanamycin, were investigated. Exposure to rubratoxin B for 24h induced secretion of each CCR5 chemokine, although the effect on CCL5 secretion was modest, and it enhanced secretion of proinflammatory cytokines tumor necrosis factor-α, CXCL8, and CCL2. Concomitant treatment with radicicol abolished the rubratoxin-induced secretion of all cytokines investigated. Geldanamycin antagonized the rubratoxin B-induced effects on CCL3 and CCL5, but not CCL4; the effects of geldanamycin were less than that of radicicol. Taken together, the results suggest that rubratoxin B, with the contribution of Hsp90, induces secretion of CCR5 chemokines.

Detection of N-(1-deoxy-D-fructos-1-yl) Fumonisins B2 and B3 in Corn by High-Resolution LC-Orbitrap MS.

The existence of glucose conjugates of fumonisin B2 (FB2) and fumonisin B3 (FB3) in corn powder was confirmed for the first time. These "bound-fumonisins" (FB2 and FB3 bound to glucose) were identified as N-(1-deoxy-D-fructos-1-yl) fumonisin B2 (NDfrc-FB2) and N-(1-deoxy-D-fructos-1-yl) fumonisin B3 (NDfrc-FB3) respectively, based on the accurate mass measurements of characteristic ions and fragmentation patterns using high-resolution liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap MS) analysis. Treatment on NDfrc-FB2 and NDfrc-FB3 with the o-phthalaldehyde (OPA) reagent also supported that D-glucose binding to FB2 and FB3 molecules occurred to their primary amine residues.

Detection of type A trichothecene di-glucosides produced in corn by high-resolution liquid chromatography-Orbitrap mass spectrometry.

The existence of di-glucosylated derivative of T-2 toxin in plant (corn powder) was confirmed for the first time in addition to that of HT-2 toxin. These masked mycotoxins (mycotoxin glucosides) were identified as T-2 toxin-di-glucoside (T2GlcGlc) and HT-2 toxin-di-glucoside (HT2GlcGlc) based on accurate mass measurements of characteristic ions and fragmentation patterns using high-resolution liquid chromatography-Orbitrap mass spectrometric (LC-Orbitrap MS) analysis. Although the absolute structure of T2GlcGlc was not clarified, two glucose molecules were suggested to be conjugated at 3-OH position in tandem when considering the structure of T-2 toxin. On the other hand, the specification of the structure seems to be more complicated in the case of HT2GlcGlc, since HT-2 toxin has two possible positions (at 3-OH and 4-OH) to be glusocylated. In addition, 15-monoacetoxyscirpenol-glucoside (MASGlc) was also detected in the identical sample.

Opposite effects of two trichothecene mycotoxins, deoxynivalenol and nivalenol, on the levels of macrophage inflammatory protein (MIP)-1α and MIP-1ß in HL60 cells.

To elucidate the mechanisms underlying the toxicities of the trichothecene mycotoxins deoxynivalenol and nivalenol, their effects on the secretion of anti-hematopoietic chemokines, macrophage inflammatory protein-1α (MIP-1α) and MIP-1ß in human promyelocytic leukemia cell line HL60 were investigated. Exposure to deoxynivalenol for 24h significantly induced the secretion of chemokines. The induction of these chemokines may account for the leukopenia after exposure to trichothecene mycotoxins. Treatment with nivalenol decreased the secretion of these chemokines. Our finding that deoxynivalenol induces the secretion of these chemokines, whereas nivalenol has the opposite effect, clearly indicates that the toxicity mechanisms of deoxynivalenol and nivalenol differ.

Rubratoxin B induces signs of fatty acid oxidation disorders (FAODs) in mice.

Rubratoxin B is a mycotoxin that causes hypoglycemia and fatty liver. We investigated the effect of rubratoxin B on hepatic glycogen content and regulation, because blood glucose levels are associated with hepatic glycogen storage. Mice were treated with 1.5mg/kg rubratoxin B for 24h. Stomachs of treated mice became extremely swollen, and the contents were significantly heavier than those of controls. Hypoglycemia stimulates appetite; therefore, rubratoxin B may perturb satiation. Rubratoxin B evidently depleted hepatic glycogen stores. Phosphoenolpyruvate carboxykinase (PEPCK) activity and mRNA levels in treated mice were reduced, indicating that rubratoxin B caused hepatic glycogen depletion by inhibiting PEPCK. PEPCK activity and mRNA levels were reduced to similar degrees; it appears that PEPCK activity is regulated transcriptionally. Levels of the PEPCK gene trans-activators phospho-CREB (active form) and C/EBPα were significantly reduced in the livers of treated mice, suggesting that these factors are important for PEPCK gene transcription. Rubratoxicosis and fatty acid oxidation disorders (FAODs) share characteristic signs, such as robust appetite, hypoglycemia, hepatic glycogen depletion, and fatty liver. Although FAODs are generally considered genetic deficiencies, our results indicate that a chemical can also cause FAOD-like signs in mice.

Nuclear factor-κB inhibitors alleviate nivalenol-induced cytotoxicity in HL60 cells.

Tricothecene mycotoxins, such as nivalenol, are toxic to leukocytes. To elucidate the molecular mechanism of nivalenol toxicity, we investigated the involvement of nuclear factor-κB (NF-κB) in nivalenol-induced cytotoxicity in HL60 cells using the NF-κB inhibitors pyrrolidinedithiocarbamate (PDTC) and dexamethasone. Cells were treated with the chemicals for 24h before assays were performed. Nivalenol elicited interleukin (IL)-8 secretion. IL-8 secretion was lower in cells concomitantly treated with nivalenol and NF-κB inhibitors than with nivalenol alone. Nivalenol reduced monocyte chemotactic protein (MCP)-1 secretion. MCP-1 secretion was higher in cells concomitantly treated with nivalenol and NF-κB inhibitors than with nivalenol alone. NF-κB inhibitors thus alleviated the effects of nivalenol, indicating that NF-κB is important for nivalenol-caused changes in cytokine secretion. Nivalenol hindered cell proliferation, and dexamethasone reduced this effect, suggesting that NF-κB contributes to cell proliferation. Thus, it appears that NF-κB is involved in nivalenol-induced toxicity in HL60 cells.

Distinct distribution of deoxynivalenol, nivalenol, and ergosterol in Fusarium-infected Japanese soft red winter wheat milling fractions.

The occurrence of mycotoxins in small grain cereals and their retention in final products are serious concerns for food safety. Previously, we investigated the fate of deoxynivalenol and nivalenol in a Japanese soft red winter wheat cultivar during milling and we found that deoxynivalenol and/or nivalenol was readily distributed among flours for human consumption. In the present study, we analyzed the ergosterol concentrations in the milling fractions as an index of fungal biomass to elucidate the relationship between deoxynivalenol/nivalenol accumulation and fungal invasion into the grain, after the in-house validation of an analytical method for quantifying ergosterol in the resulting milling fractions (patent flour, low-grade flour, bran, and shorts). Using three samples with different levels of deoxynivalenol and/or nivalenol contamination, the contents of deoxynivalenol/nivalenol and ergosterol in the resulting milling fractions were evaluated. The concentration of ergosterol was always lowest in patent flour and highest in bran or shorts, indicating that most of the fungi is retained in the outer layers of grain (bran and shorts) even in highly contaminated grain. On the other hand, the concentrations of deoxynivalenol and nivalenol were similar in the low-grade and patent flours and only slightly lower than in the medium-level and high-level contaminated grains. Moreover, the percentage distribution of ergosterol was higher in bran than in other fractions in all cases, which differed from that of deoxynivalenol/nivalenol. This result indicates the diffusion of deoxynivalenol/nivalenol inside the grain that is independent of fungal invasion.

Distribution of deoxynivalenol and nivalenol in milling fractions from fusarium-infected Japanese wheat cultivars.

The fate of the Fusarium mycotoxins deoxynivalenol and nivalenol during the milling of Japanese wheat cultivars artificially infected with Fusarium was investigated. Grain samples with different mycotoxin concentrations were milled using a laboratory-scale test mill to produce eight fractions: three breaking flours (1B, 2B, and 3B), three reduction flours (1M, 2M, and 3M), wheat bran, and wheat shorts. Patent flour for human consumption was made from the 1B, 2B, 1M, and 2M flours, and low-grade flour was made from 3B and 3M flours. The four resulting samples (patent flour, low-grade flour, bran, and shorts) were analyzed for deoxynivalenol and/or nivalenol with an in-house validated analytical method using high-performance liquid chromatography with UV absorbance detection. In samples with different mycotoxin concentrations, the distribution of those toxins differed among the milling fractions. Grains with a lower level of contamination produced bran and shorts samples with a high relative concentration of nivalenol. A high percentage of nivalenol was found in patent flour, followed by bran. Contrary to the less-contaminated sample, the concentration of nivalenol in moderately contaminated grain was high only in the shorts sample. The highest percentage of deoxynivalenol and nivalenol was observed in the patent flour. The results of this study indicate that the distribution of deoxynivalenol and nivalenol in milled Japanese wheat could be influenced by the contamination level of the original grain, and the milling process is not always effective for removal of toxins from wheat grains.

Limited surveillance of fumonisins in brown rice and wheat harvested in Japan.

Fumonisins are mycotoxins mainly produced by Fusarium verticillioides, which is a major contaminant of corn. However, there are sporadic reports of fumonisin contamination in wheat worldwide. The rice adherent fungus Gibberella fujikuroi is taxonomically closely related to F. verticillioides. Therefore, the potential risk of fumonisin contamination in rice and wheat is significant. Previously, a sensitive detection method utilizing liquid chromatography with tandem electrospray mass spectrometry (LC-ESI-MS-MS) was developed for the determination of fumonisins in brown rice. In the present study, the incidence of fumonisins in brown rice and wheat harvested in Japan was investigated using LC-ESI-MS-MS. Forty-eight rice samples and 47 wheat samples were screened and analyzed for the major B-type fumonisins: fumonisin B1 (FB1) and fumonisin B2 (FB2). About 1 kg of rice or wheat seed was divided into three subsamples, and 10 g from each subsample was used for the analysis. The limits of detection were 0.012 and 0.011 mg/kg for FBt and FB2, respectively, in rice samples and 0.010 and 0.008 mg/kg for FB1 and FB2, respectively, in wheat samples. The mean (standard deviation) recoveries of FB1 spiked at 0.50 mg/kg into toxin-free rice and wheat samples were 77.6 (4.2)% and 84.5 (3.1)%, respectively. One of the wheat samples was positive for FBt with a value greater than the limit of detection,but no fumonisin was found in any of the rice samples. This is the first report of fumonisins detected in Japanese wheat.

Rubratoxin B induces interleukin-6 secretion in mouse white adipose tissues and 3T3-L1 adipocytes.

Rubratoxin B is a mycotoxin that causes hepatic fatty changes. We examined whether white adipose tissue (WAT) contributes to rubratoxin B toxicity through effects on interleukin (IL)-6. Rubratoxin B was intraperitoneally injected into mice at 1.5mg/kg. Urinary albumin and macrophage inflammatory protein (MIP)-2 secretion were increased 24h after treatment with rubratoxin B. Rubratoxin B was previously reported to induce IL-6 secretion, although the secreting tissue was unknown. Here, rubratoxin B prominently augmented IL-6 transcription in epididymal WAT and to a lesser extent in perirenal WAT and liver. Rubratoxin B may thus exert its toxicity partly through IL-6 secretion from WATs. In contrast, MIP-2 gene expression increased only in liver. To examine the specific involvement of adipocytes, we used mouse 3T3-L1 cells, an in vitro differentiation model of adipocytes. Expression of IL-6 and MIP-2 mRNA in 3T3-L1 adipocytes after 24h of rubratoxin B treatment increased dose-dependently. Rubratoxin B also increased IL-6 and MIP-2 secretion from 3T3-L1 adipocytes. The increase in IL-6 secretion was markedly higher than the increase in IL-6 gene transcription, indicating that rubratoxin B-induced secretion of IL-6 from 3T3-L1 adipocytes is chiefly controlled post-transcriptionally. Rubratoxin B is thus the first mycotoxin known to exert its toxicity through effects on WATs.

Induced secretion of insulin-like growth factor binding protein-1 (IGFBP-1) in human hepatoma cell HepG2 by rubratoxin B.

The induction of insulin-like growth factor binding protein-1 (IGFBP-1) secretion by rubratoxin B was investigated using human hepatoma cell line HepG2; we also documented the involvement of stress-activated MAP kinases [c-Jun-N-terminal kinases (JNKs) and p38s] in this process. Rubratoxin B dramatically enhanced IGFBP-1 secretion, which peaked at a concentration of 40 microg/ml. The amount of IGFBP-1 mRNA increased with time and plateaued at 6 h. Compared with the amounts of IGFBP-1 secreted, the induction ratios of transcription were much smaller, indicating that IGFBP-1 secretion is regulated chiefly post-transcriptionally. The result of concomitant treatment with rubratoxin B and JNK inhibitor indicated that JNKs do not affect rubratoxin B-induced IGFBP-1 secretion. Alternatively, rubratoxin B-associated induction of IGFBP-1 secretion was marked in the absence of p38 inhibitor but attenuated in its presence. Therefore, p38s appear to stimulate rubratoxin B-induced IGFBP-1 secretion. Treatment with p38 inhibitor slightly increased the amount of rubratoxin B-induced IGFBP-1 mRNA. However this induction ratio was smaller than that of rubratoxin B-induced secretion, suggesting that p38s regulate IGFBP-1 secretion both transcriptionally and post-transcriptionally. In this study, we showed that rubratoxin B induces IGFBP-1 levels in HepG2 cells and p38s contribute to this process.

Stress-activated MAP kinases regulate rubratoxin B-caused cytotoxicity and cytokine secretion in hepatocyte-derived HepG2 cells.

The effects of stress-activated MAP kinases (SAPKs) on biological phenomena in HepG2 cells caused by the hepatotoxin rubratoxin B were investigated. The amounts of phosphorylated (active) SAPKs (c-Jun N-terminal kinases (JNKs) and p38s) were significantly increased after treating cells with rubratoxin B, suggesting that rubratoxin B exerts its toxicity through SAPK signal transduction pathways. Compared with rubratoxin B-treatment alone, treatment with both rubratoxin B and the JNK inhibitor SP600125 decreased cell morphology changes and the activity of the apoptosis-related enzymes caspase-3 and caspase-7, indicating that JNKs are involved in rubratoxin B-induced apoptosis. The p38 inhibitor SB203580 had the same general effects as SP600125; however, its effects were rather weak. The percent inhibition of cell proliferation by SAPKs were nearly the same with or without rubratoxin B, suggesting that the regulation of SAPKs is independent of rubratoxin B effects. SAPK inhibitors decreased rubratoxin B-induced secretion of interleukin-8 and macrophage colony stimulating factor; SP600125 impaired rubratoxin B-induced granulocyte-macrophage colony stimulating factor secretion, but SB203580 enhanced this secretion. The effects of SAPK inhibitors on the levels of cytokine mRNAs showed basically the same pattern as their effects on cytokine secretion, except that their relative effects on mRNA levels was smaller. Thus, SAPKs play important roles in rubratoxin B-induced cytokine secretion, mainly post-transcriptionally.

Rubratoxin B induced the secretion of hepatic injury-related colony stimulating factors in human hepatoma cells.

The induction of cytokine secretion by rubratoxin B was investigated using human hepatoma cell lines HepG2 and HuH-7. Interleukin (IL)-8, macrophage colony stimulating factor (M-CSF) and granulocyte-macrophage (GM)-CSF were detected in the media of rubratoxin B-treated both cell lines, and their levels peaked at about 40 microg/ml. Rubratoxin B-induced cytokine secretion was enhanced by tumor necrosis factor (TNF)-alpha in HepG2 cells. While emodin increased GM-CSF secretion, the secretion of the others was decreased, indicating that they are regulated differently in rubratoxin B-treated HepG2 cells. To our knowledge, this is the first report that an exogenous stimulus induced the secretion of M-CSF and GM-CSF in hepatocyte-derived hepatoma cells, suggesting that rubratoxin B is an excellent model compound to study the mechanisms of M-CSF and GM-CSF secretion. Our results showed that hepatotoxin rubratoxin B has a potential to induce the secretion of these two CSFs, implying that these factors play roles in rubratoxin B-caused hepatic injury.

Suppression of cytotoxin-induced cell death in isolated hepatocytes by tea catechins.

To elucidate the hepatoprotective effects of green tea catechins, the following experiments were conducted utilizing (-)-epigallocatechin-3-gallate (EGCG), the major component of green tea catechin, together with other catechins. The protective effects of catechins against hepatotoxins, bromobenzene or rubratoxin B, were examined in primary cultures of rat hepatocytes. Bromobenzene and rubratoxin B are known to induce necrosis and apoptosis of cells, respectively. After 24-h treatment with toxin, EGCG and (-)-epigallocatechin-3-(3"-O-methyl)gallate (EGCg-3"-OMe) suppressed the bromobenzene-induced morphological change and dose-dependently prevented bromobenzene-induced cell death. Both catechins also prevented apoptotic cell death caused by rubratoxin B. In rubratoxin B-treated cells, both catechins were found to suppress the activation of caspase-3 by rubratoxin B. The results in the present study suggest that EGCG and EGCg-3"-OMe are potent hepatoprotective agents. This report is the first to show that catechins suppress cytotoxin-induced cell death.

Enhancing effect of zinc on hepatoprotectivity of epigallocatechin gallate in isolated rat hepatocytes.

The influence of metal ions (Fe2+, Cu2+, Zn2+) on the hepatoprotective activity of epigallocatechin gallate (EGCG) against hepatotoxin-induced cell injury was investigated. Primary cultures of rat hepatocytes were treated with a well-known hepatotoxin, bromobenzene (BB), in the presence of EGCG only or EGCG plus each metal ion. After 24 h, 0.02 mM EGCG did not show protective activity on the cultured hepatocytes. In contrast, the hepatocytes were protected against BB in the presence of 0.02 mM EGCG and 0.02 mM zinc. The addition of only zinc could not protect hepatocytes against BB. These results suggest that the formation of the zinc-EGCG complex is very important in the enhancement of the hepatoprotective activity of EGCG. The complexation of EGCG with zinc was confirmed by UV-VIS absorption spectroscopy.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced down-regulation of glucose transporting activities in mouse 3T3-L1 preadipocyte.

The possibility that 3T3-L1 preadipocytes, while the level of its glucose uptake activity is relatively low, may offer a useful tool for studying the cause for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced "lipolytic response" was studied. It was established first, that TCDD causes reduction of glucose uptake, one of the hallmark events of the lipolytic process. Then the function of c-Src was investigated. The antisense c-src oligonucleotide decreased the inhibitory action of TCDD on glucose uptake activity in a sequence specific manner. Since antisense oligonucleotides are known to own their blocking effects to their ability to reduce translation of proteins, Western blotting analysis was performed to verify their effectiveness. As expected, the treatment of pre-adipocytes with antisense c-src oligonucleotide reduced c-Src in a sequence specific manner. The treatment of antisense c-src oligonucleotide alone was sufficient to diminish the inhibitory action of TCDD on glucose uptake activity in 3T3-L1 cells, indicating that c-Src is somehow involved in the action of TCDD. In a similar manner, the contribution of c-Fos was investigated using antisense c-fos oligonucleotide, since c-Fos is known to be one of the most affected proteins by c-Src activation among AP-1 members. The treatment of antisense c-fos oligonucleotide did not block the effect of TCDD on glucose uptake activity in 3T3-L1 cells. Therefore, it is unlikely that c-Fos is very important in the lipolytic signal transduction of TCDD mediated through c-Src. In order to determine the relationship between c-Src and c-Myc in the mitotic signal transduction pathway, the effect of antisense c-myc oligonucleotide was investigated. Basically the same result as antisense c-src oligonucleotide experiment was obtained thereby, suggesting the importance of c-Myc as well as c-Src in the signal transduction of TCDD. To show the effect of antisense c-myc oligonucleotide treatment, the level of c-Myc protein by Western blotting and electrophoretic gel-mobility shift assay was assessed. However, antisense c-myc oligonucleotide treatment increased the activity of c-Myc in a sequence specific manner. This may be the result of cellular compensatory response to the initial suppression of c-Myc by antisense treatment. The observation that antisense c-fos oligonucleotide could not block the effect of TCDD indicates that this preadipocyte model is different from the adipocyte differentiation model.

Effects of src-deficiency on the expression of in vivo toxicity of TCDD in a strain of c-src knockout mice procured through six generations of backcrossings to C57BL/6 mice.

The effect of TCDD was studied in c-src-deficient C57BL6-src(tm1sor) (N6 src -/- and -/+) mice, and their wild-type littermate mice (N6 src +/+). The former was created from the original strain of B6, 129-src(tm1sor) mice through six generations of backcrossings with C57BL6 mice. The results of a high dose TCDD toxicity tests in male mice indicated that N6 src-/+ mice were significantly less responsive to the toxic action of TCDD (115 microg/kg single i.p. injection) than N6 src+/+ mice in terms of reduced % body weight gain, the increase in the liver to body weight ratio, and the decrease in the adipose tissue to liver weight ratio and in the weight of pancreas. To understand the cause for these differential effects of TCDD we studied TCDD-induced changes in several biochemical parameters at day 10 and found that most drastically affected ones were glycogen depletion and phosphoenolpyruvate carboxykinase (PEPCK) downregulation. In addition, the degree of triglyceride accumulation in liver was less pronounced in N6-/+ than in N6+/+ mice. These findings suggest that the absence of c-src expression indeed affects the development of selected, TCDD-induced toxic endpoints that are related to wasting syndrome.