Contributions of the anaplerotic reaction enzymes pyruvate carboxylase and phosphoenolpyruvate carboxylase to l-lysine production in Corynebacterium glutamicum.

Anaplerotic reactions catalyzed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) have important roles in the production of l-lysine to replenish oxaloacetic acid (OAA) in Corynebacterium glutamicum. However, the relative contributions of these enzymes to l-lysine production in C. glutamicum are not fully understood. In this study, using a parent strain (P) carrying a feedback inhibition-resistant aspartokinase with the T311I mutation, we constructed a PC gene-deleted mutant strain (PΔPC) and a PEPC gene-deleted mutant strain (PΔPEPC). Although the growth of both mutant strains was comparable to the growth of strain P, the maximum l-lysine production in strains PΔPC and PΔPEPC decreased by 14% and 49%, respectively, indicating that PEPC strongly contributed to OAA supply. l-Lysine production in strain PΔPC slightly decreased during the logarithmic phase, while production during the early stationary phase was comparable to production in strain P. By contrast, strain PΔPEPC produced l-lysine in an amount comparable to the production of strain P during the logarithmic phase; l-lysine production after the early stationary phase was completely stopped in strain PΔPEPC. These results indicate that OAA is supplied by both PC and PEPC during the logarithmic phase, while only PEPC can continuously supply OAA after the logarithmic phase.

Methionine utilization by bifidobacteria: possible existence of a reverse transsulfuration pathway.

Although bifidobacteria are already widely used as beneficial microbes with health-promoting effects, their amino acid utilization and metabolism are not yet fully understood. Knowledge about the metabolism of sulfur-containing amino acids in bifidobacteria is especially limited. In this study, we tested the methionine utilization ability of several bifidobacterial strains when it was the sole available sulfur source. Although bifidobacteria have long been predominantly considered to be cysteine auxotrophs, we showed that this is not necessarily the case.

Engineering of Corynebacterium glutamicum as a prototrophic pyruvate-producing strain: Characterization of a ramA-deficient mutant and its application for metabolic engineering.

To construct a prototrophic Corynebacterium glutamicum strain that efficiently produces pyruvate from glucose, the effects of inactivating RamA, a global regulator responsible for activating the oxidative tricarboxylic acid (TCA) cycle, on glucose metabolism were investigated. ΔramA showed an increased specific glucose consumption rate, decreased growth, comparable pyruvate production, higher formation of lactate and acetate, and lower accumulation of succinate and 2-oxoglutarate compared to the wild type. A significant decrease in pyruvate dehydrogenase complex activity was observed for ΔramA, indicating reduced carbon flow to the TCA cycle in ΔramA. To create an efficient pyruvate producer, the ramA gene was deleted in a strain lacking the genes involved in all known lactate- and acetate-producing pathways. The resulting mutant produced 161 mM pyruvate from 222 mM glucose, which was significantly higher than that of the parent (89.3 mM; 1.80-fold).

Exchanging Alkyl Groups through Unstrained C-C Bond Cleavage in the Presence of a Copper Catalyst.

Although numerous reports exist on strained C-C bond cleavage reactions in aryl substitutions, the cleavage methodology for unstrained C-C bonds in alkylation reactions has not yet been established. We found that unstrained allylic C-C bonds can be cleaved using α-radicals to form C(sp3 )-C(sp3 ) bonds in the presence of a copper catalyst. In this reaction, the property of leaving and loading radicals is very important for radical fragmentations. In this paper, we investigated the effects of these properties in cleavage reactions for unstrained C-C bonds.

Isolation and amino acid sequence of a dehydratase acting on d-erythro-3-hydroxyaspartate from Pseudomonas sp. N99, and its application in the production of optically active 3-hydroxyaspartate.

An enzyme catalyzing the ammonia-lyase reaction for the conversion of d-erythro-3-hydroxyaspartate to oxaloacetate was purified from the cell-free extract of a soil-isolated bacterium Pseudomonas sp. N99. The enzyme exhibited ammonia-lyase activity toward l-threo-3-hydroxyaspartate and d-erythro-3-hydroxyaspartate, but not toward other 3-hydroxyaspartate isomers. The deduced amino acid sequence of the enzyme, which belongs to the serine/threonine dehydratase family, shows similarity to the sequence of l-threo-3-hydroxyaspartate ammonia-lyase (EC 4.3.1.16) from Pseudomonas sp. T62 (74%) and Saccharomyces cerevisiae (64%) and serine racemase from Schizosaccharomyces pombe (65%). These results suggest that the enzyme is similar to l-threo-3-hydroxyaspartate ammonia-lyase from Pseudomonas sp. T62, which does not act on d-erythro-3-hydroxyaspartate. We also then used the recombinant enzyme expressed in Escherichia coli to produce optically pure l-erythro-3-hydroxyaspartate and d-threo-3-hydroxyaspartate from the corresponding dl-racemic mixtures. The enzymatic resolution reported here is one of the simplest and the first enzymatic method that can be used for obtaining optically pure l-erythro-3-hydroxyaspartate.

Pyruvate dehydrogenase complex regulator (PdhR) gene deletion boosts glucose metabolism in Escherichia coli under oxygen-limited culture conditions.

Pyruvate dehydrogenase complex regulator (PdhR) is a transcriptional regulator that negatively regulates formation of pyruvate dehydrogenase complex (PDHc), NADH dehydrogenase (NDH)-2, and cytochrome bo3 oxidase in Escherichia coli. To investigate the effects of a PdhR defect on glucose metabolism, a pdhR deletion mutant was derived from the wild-type E. coli W1485 strain by λ Red-mediated recombination. While no difference in the fermentation profiles was observed between the two strains under oxygen-sufficient conditions, under oxygen-limited conditions, the growth level of the wild-type strain was significantly decreased with retarded glucose consumption accompanied by by-production of substantial amounts of pyruvic acid and acetic acid. In contrast, the mutant grew and consumed glucose more efficiently than did the wild-type strain with enhanced respiration, little by-production of pyruvic acid, less production yield and rates of acetic acid, thus displaying robust metabolic activity. As expected, increased activities of PDHc and NDH-2 were observed in the mutant. The increased activity of PDHc may explain the loss of pyruvic acid by-production, probably leading to decreased acetic acid formation, and the increased activity of NDH-2 may explain the enhanced respiration. Measurement of the intracellular NAD+/NADH ratio in the mutant revealed more oxidative or more reductive intracellular environments than those in the wild-type strain under oxygen-sufficient and -limited conditions, respectively, suggesting another role of PdhR: maintaining redox balance in E. coli. The overall results demonstrate the biotechnological advantages of pdhR deletion in boosting glucose metabolism and also improve our understanding of the role of PdhR in bacterial physiology.

Boosting Anaplerotic Reactions by Pyruvate Kinase Gene Deletion and Phosphoenolpyruvate Carboxylase Desensitization for Glutamic Acid and Lysine Production in Corynebacterium glutamicum.

In the 1980s, Shiio and coworkers demonstrated using random mutagenesis that the following three phenotypes were effective for boosting lysine production by Corynebacterium glutamicum: (1) low-activity-level citrate synthase (CSL), (2) phosphoenolpyruvate carboxylase (PEPC) resistant to feedback inhibition by aspartic acid (PEPCR), and (3) pyruvate kinase (PYK) deficiency. Here, we reevaluated these phenotypes and their interrelationship in lysine production using recombinant DNA techniques.The pyk deletion and PEPCR (D299N in ppc) independently showed marginal effects on lysine production, but both phenotypes synergistically increased lysine yield, demonstrating the importance of PEPC as an anaplerotic enzyme in lysine production. Similar effects were also found for glutamic acid production. CSL (S252C in gltA) further increased lysine yield. Thus, using molecular techniques, the combination of these three phenotypes was reconfirmed to be effective for lysine production. However, a simple CSL mutant showed instabilities in growth and lysine yield.Surprisingly, the pyk deletion was found to increase biomass production in wild-type C. glutamicum ATCC13032 under biotin-sufficient conditions. The mutant showed a 37% increase in growth (based on OD660) compared with the ATCC13032 strain in a complex medium containing 100 g/L glucose. Metabolome analysis revealed the intracellular accumulation of excess precursor metabolites. Thus, their conversion into biomass was considered to relieve the metabolic distortion in the pyk-deleted mutant. Detailed physiological studies of various pyk-deleted mutants also suggested that malate:quinone oxidoreductase (MQO) is important to control both the intracellular oxaloacetic acid (OAA) level and respiration rate. These findings may facilitate the rational use of C. glutamicum in fermentation industries.

Pyruvate kinase deletion as an effective phenotype to enhance lysine production in Corynebacterium glutamicum ATCC13032: Redirecting the carbon flow to a precursor metabolite.

Various attempts have been made to enhance lysine production in Corynebacterium glutamicum. Pyruvate kinase (PYK) defect is one of the strategies used to enhance the supply of oxaloacetic acid (OAA), a precursor metabolite for lysine biosynthesis. However, inconsistent effects of this mutation have been reported: positive effects of PYK defect in mutants having phosphoenolpyruvate carboxylase (PEPC) desensitized to feedback inhibition by aspartic acid, while negative effects in simple PYK gene (pyk) knockout mutants. To address these discrepancies, the effects of pyk deletion on lysine yield were investigated with or without the D299N mutation in ppc rendering PEPC desensitization. C. glutamicum ATCC13032 mutant strain P with a feedback inhibition-desensitized aspartokinase was used as the parent strain, producing 9.36 g/L lysine from 100 g/L glucose in a jar fermentor culture. Under these conditions, while the simple mutant D2 with pyk deletion or R2 with the PEPC-desensitization mutation showed marginally increased lysine yield (∼1.1-fold, not significant), the mutant DR2 strain having both mutations showed synergistically increased lysine productivity (1.38-fold, 12.9 g/L). Therefore, the pyk deletion is effective under a PEPC-desensitized background, which ensures enhanced supply of OAA, thus clarifying the discrepancies. A citrate synthase defective mutation (S252C in gltA) further increased the lysine yield in strain DR2 (1.68-fold, 15.7 g/L). Thus, these three mutations coordinately enhanced the lysine yield. Both the malate:quinone oxidoreductase activity and respiration rate were significantly reduced in strains D2 and DR2. Overall, these results provide valuable knowledge for engineering the anaplerotic reaction to increase lysine yield in C. glutamicum.

Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.

Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum.

A Copper-Catalyzed Formal [3 + 2]-Cycloaddition for the Synthesis of All Different Aryl-Substituted Furans and Thiophenes.

A highly efficient formal [3 + 2]-cycloaddition was established using a copper catalyst. The resulting dihydrofurans were subjected to oxidation followed by arylations to produce tetraarylfurans. In addition, the dihydrofuran can be converted to diaryldihydrothiophene by using Lawesson's reagent. This protocol will facilitate the synthesis of all different aryl-substituted furans and thiophenes.

Structural insights into the substrate stereospecificity of D-threo-3-hydroxyaspartate dehydratase from Delftia sp. HT23: a useful enzyme for the synthesis of optically pure L-threo- and D-erythro-3-hydroxyaspartate.

D-threo-3-Hydroxyaspartate dehydratase (D-THA DH) is a fold-type III pyridoxal 5'-phosphate-dependent enzyme, isolated from a soil bacterium of Delftia sp. HT23. It catalyzes the dehydration of D-threo-3-hydroxyaspartate (D-THA) and L-erythro-3-hydroxyaspartate (L-EHA). To elucidate the mechanism of substrate stereospecificity, crystal structures of D-THA DH were determined in complex with various ligands, such as an inhibitor (D-erythro-3-hydroxyaspartate (D-EHA)), a substrate (L-EHA), and the reaction intermediate (2-amino maleic acid). The C (ß) -OH of L-EHA occupied a position close to the active-site Mg(2+), clearly indicating a possibility of metal-assisted C (ß) -OH elimination from the substrate. In contrast, the C (ß) -OH of an inhibitor was bound far from the active-site Mg(2+). This suggests that the substrate specificity of D-THA DH is determined by the orientation of the C (ß) -OH at the active site, whose spatial arrangement is compatible with the 3R configuration of 3-hydroxyaspartate. We also report an optically pure synthesis of L-threo-3-hydroxyaspartate (L-THA) and D-EHA, promising intermediates for the synthesis of ß-benzyloxyaspartate, by using a purified D-THA DH as a biocatalyst for the resolution of racemic DL-threo-3-hydroxyaspartate (DL-THA) and DL-erythro-3-hydroxyaspartate (DL-EHA). Considering 50 % of the theoretical maximum, efficient yields of L-THA (38.9 %) and D-EHA (48.9 %) as isolated crystals were achieved with >99 % enantiomeric excess (e.e.). The results of nuclear magnetic resonance signals verified the chemical purity of the products. We were directly able to isolate analytically pure compounds by the recrystallization of acidified reaction mixtures (pH 2.0) and thus avoiding the use of environmentally harmful organic solvents for the chromatographic purification.

Effect of pyruvate kinase gene deletion on the physiology of Corynebacterium glutamicum ATCC13032 under biotin-sufficient non-glutamate-producing conditions: Enhanced biomass production.

The effect of pyruvate kinase gene (pyk) deletion on the physiology of Corynebacterium glutamicum ATCC13032 was investigated under biotin-sufficient, non-glutamate-producing conditions. In a complex medium containing 100 g/L glucose, a defined pyk deletion mutant, strain D1, exhibited 35% enhancement in glucose consumption rate, 37% increased growth and a 57% reduction in respiration rate compared to the wild-type parent. Significant upregulation of phosphoenolpyruvate (PEP) carboxylase and downregulation of PEP carboxykinase activities were observed in the D1 mutant, which may have prevented over-accumulation of PEP caused by the pyk deletion. Moreover, we found a dramatic 63% reduction in the activity of malate:quinone oxidoreductase (MQO) in the D1 mutant. MQO, a TCA cycle enzyme that converts malate to oxaloacetate (OAA), constitutes a major primary gate to the respiratory chain in C. glutamicum, thus explaining the reduced respiration rate in the mutant. Additionally, pyruvate carboxylase gene expression was downregulated in the mutant. These changes seemed to prevent OAA over-accumulation caused by the activity changes of PEP carboxylase/PEP carboxykinase. Intrinsically the same alterations were observed in the cultures conducted in a minimal medium containing 20 g/L glucose. Despite these responses in the mutant, metabolic distortion caused by pyk deletion under non-glutamate-producing conditions required amelioration by increased biomass production, as metabolome analysis revealed increased intracellular concentrations of several precursor metabolites for building block formation associated with pyk deletion. These fermentation profiles and metabolic alterations observed in the mutant reverted completely to the wild-type phenotypes in the pyk-complemented strain, suggesting the observed metabolic changes were caused by the pyk deletion. These results demonstrated multilateral strategies to overcome metabolic disturbance caused by pyk deletion in this bacterium.

Metabolic flux redirection from a central metabolic pathway toward a synthetic pathway using a metabolic toggle switch.

Overexpression of genes in production pathways and permanent knockout of genes in competing pathways are often employed to improve production titer and yield in metabolic engineering. However, the deletion of a pathway responsible for growth and cell maintenance has not previously been employed, even if its competition with the production pathway is obvious. In order to optimize intracellular metabolism at each fermentation phase for bacterial growth and production, a methodology employing conditional knockout is required. We constructed a metabolic toggle switch in Escherichia coli as a novel conditional knockout approach and applied it to isopropanol production. The resulting redirection of excess carbon flux caused by interruption of the TCA cycle via switching gltA OFF improved isopropanol production titer and yield up to 3.7 and 3.1 times, respectively. This approach is a useful tool to redirect carbon flux responsible for bacterial growth and/or cell maintenance toward a synthetic production pathway.

Crystallization and preliminary X-ray diffraction studies of D-threo-3-hydroxyaspartate dehydratase isolated from Delftia sp. HT23.

D-threo-3-Hydroxyaspartate dehydratase (D-THA DH) isolated from the soil bacterium Delftia sp. HT23 is a novel enzyme consisting of 380 amino-acid residues which catalyzes the conversion of D-threo-3-hydroxyaspartate to oxaloacetate and ammonia. D-THA DH also catalyzes the dehydration of L-threo-3-hydroxyaspartate, L-erythro-3-hydroxyaspartate and D-serine. The amino-acid sequence of D-THA DH shows significant similarity to that of two eukaryotic D-serine dehydratases derived from Saccharomyces cerevisiae and chicken kidney. D-THA DH is classified into the fold-type III group of pyridoxal enzymes and is the first example of a fold-type III dehydratase derived from a prokaryote. Overexpression of recombinant D-THA DH was carried out using a Rhodococcus erythropolis expression system and the obtained protein was subsequently purified and crystallized. The crystals of D-THA DH belonged to space group I4122, with unit-cell parameters a=b=157.3, c=157.9 Å. Single-wavelength anomalous diffraction data were collected to a resolution of 2.0 Šusing synchrotron radiation at the wavelength of the Br K absorption edge.

Contribution of the 7ß-hydroxysteroid dehydrogenase from Ruminococcus gnavus N53 to ursodeoxycholic acid formation in the human colon.

Bile acid composition in the colon is determined by bile acid flow in the intestines, the population of bile acid-converting bacteria, and the properties of the responsible bacterial enzymes. Ursodeoxycholic acid (UDCA) is regarded as a chemopreventive beneficial bile acid due to its low hydrophobicity. However, it is a minor constituent of human bile acids. Here, we characterized an UDCA-producing bacterium, N53, isolated from human feces. 16S rDNA sequence analysis identified this isolate as Ruminococcus gnavus, a novel UDCA-producer. The forward reaction that produces UDCA from 7-oxo-lithocholic acid was observed to have a growth-dependent conversion rate of 90-100% after culture in GAM broth containing 1 mM 7-oxo-lithocholic acid, while the reverse reaction was undetectable. The gene encoding 7ß-hydroxysteroid dehydrogenase (7ß-HSDH), which facilitates the UDCA-producing reaction, was cloned and overexpressed in Escherichia coli. Characterization of the purified 7ß-HSDH revealed that the kcat/Km value was about 55-fold higher for the forward reaction than for the reverse reaction, indicating that the enzyme favors the UDCA-producing reaction. As R. gnavus is a common, core bacterium of the human gut microbiota, these results suggest that this bacterium plays a pivotal role in UDCA formation in the colon.

In ovo exposure to nonylphenol and bisphenol A resulted in dose-independent feminization of male gonads in Japanese quail (Coturnix japonica) embryos.

Sex reversal effects of nonylphenol and bisphenol A on the gonads in F(1) (AWE × WE) Japanese quail (Coturnix japonica) embryos were investigated using an in vivo screening model developed previously. The F(1) (AWE × WE) Japanese quail are a useful avian model because sex differentiation is confirmed by the plumage color before hatching, ruled by a criss-cross inheritance. The nonylphenol at 200, 2,000, 20,000, and 200,000 ng/egg and bisphenol A at 20, 200, 2,000, and 20,000 ng/egg were injected into the egg white just before incubation. At 16 d of incubation, embryos were subjected to a complete necropsy, and their gonads were both grossly observed and examined histopathologically and morphometrically. Grossly, genetic sex was confirmed because plumage color coincided completely with the external sex phenotype of the gonads in all embryos. Histopathologically, feminization of the male gonad, called ovotestis, developed in the left testis in all nonylphenol- and bisphenol A-treated groups. The incidence of the lesion in all treated groups was significantly higher than that in the control group, whereas there were no dose-dependent changes in the incidence and area of the ovotestis in both nonylphenol- and bisphenol A-treated groups. The present study revealed that nonylphenol and bisphenol A have a dose-independent potential of ovotestis induction in the Japanese quail embryo.

Alterations of glucose metabolism in Escherichia coli mutants defective in respiratory-chain enzymes.

The effects of reduced efficiency of proton-motive force (pmf) generation on glucose metabolism were investigated in Escherichia coli respiratory-chain mutants. The respiratory chain of E. coli consists of two NADH dehydrogenases and three terminal oxidases, all with different abilities to generate a pmf. The genes for isozymes with the highest pmf-generating capacity (NADH dehydrogenase-1 and cytochrome bo3 oxidase) were knocked out singly or in combination, using a wild-type strain as the parent. Analyses of glucose metabolism by jar-fermentation revealed that the glucose consumption rate per cell increased with decreasing efficiency of pmf generation, as determined from the growth parameters of the mutants. The highest rate of glucose metabolism was observed in the double mutant, and the lowest was observed in the wild-type strain. The respiration rates of the single-knockout mutants were comparable to that of the wild-type strain, and that of the double mutant was higher, apparently as a result of the upregulation of the remaining respiratory chain enzymes. All of the strains excreted 2-oxoglutaric acid as a product of glucose metabolism. Additionally, all of the mutants excreted pyruvic acid and/or acetic acid. Interestingly, the double mutant excreted L-glutamic acid. Alterations of the fermentation profiles provide clues regarding the metabolic regulation in each mutant.

Mechanism of increased respiration in an H+-ATPase-defective mutant of Corynebacterium glutamicum.

We previously reported that a spontaneous H(+)-ATPase-defective mutant of Corynebacterium glutamicum, F172-8, derived from C. glutamicum ATCC 14067, showed enhanced glucose consumption and respiration rates. To investigate the genome-based mechanism of enhanced respiration rate in such C. glutamicum mutants, A-1, an H(+)-ATPase-defective mutant derived from C. glutamicum ATCC 13032, which harbors the same point mutation as F172-8, was used in this study. A-1 showed similar fermentation profiles to F172-8 when cultured in a jar fermentor. Enzyme activity measurements, quantitative real-time PCR, and DNA microarray analysis suggested that A-1 enhanced malate:quinone oxidoreductase/malate dehydrogenase and l-lactate dehydrogenase/NAD(+)-dependent-lactate dehydrogenase coupling reactions, but not NADH dehydrogenase-II, for reoxidation of the excess NADH arising from enhanced glucose consumption. A-1 also up-regulated succinate dehydrogenase, which may result in the relief of excess proton-motive force (pmf) in the H(+)-ATPase mutant. In addition, the transcriptional level of cytochrome bd oxidase, but not cytochrome bc(1)-aa(3), also increased, which may help prevent the excess pmf generation caused by enhanced respiration. These results indicate that C. glutamicum possesses intriguing strategies for coping with NADH over-accumulation. Furthermore, these mechanisms are different from those in Escherichia coli, even though the two species use similar strategies to prevent excess pmf generation.

Endocrine disrupting effects of low dose 17 ß-estradiol (E2) on the Japanese quail (Coturnix japonica) were detected by modified one-generation reproduction study.

Previously, we investigated endocrine disrupting effects of 17 ß-estradiol (E(2)) on Japanese quail (Coturnix japonica) in the avian reproduction test according to the testing guidelines, in which new endpoints such as blood vitellogenin (VTG) concentration in parent quails and pathology of F(1) chicks were added, and consequently these additional endpoints suggested to be sensitive markers for detecting any impacts of endocrine disrupting effects (Shibuya et al., 2005b). In the present study, to investigate low dose effects of estrogenic endocrine disrupting chemicals in birds, the avian reproduction study of E(2) at low dose levels was conducted using Japanese quail with additional endpoints such as observations of F(1) chicks until 10 weeks of age, histopathology of F(1) chicks at 14 days and 10 weeks of age and blood VTG concentration in parent quails. Sixteen pairs of 10-week-old quails were fed a low phytoestrogen diet containing E(2) at 0 (control), 0.3, 3, and 30 ppm for 6 weeks, and parent quails, eggs and offspring were examined. F(1) chicks were maintained up to 14 days or 10 weeks of age. Serum E(2) and VTG concentrations in males of the E(2) 3- and 30-ppm groups and in females of the E(2) 30-ppm groups were significantly elevated. In the E(2) 30-ppm group, two parent females died, and toxic changes such as suppression of body weight gain, decrease in food consumption and atrophic and degenerative changes of the reproductive organs were observed in parent quails. In the same group, the number of eggs laid and the fertility rate of eggs were significantly decreased. In addition, the viability of F(1) chicks in the E(2) 30-ppm group were significantly decreased at 10 weeks of age. On the other hand, no abnormalities described above were observed in any parent quails, eggs and F(1) chicks in the E(2) 3- and 0.3-ppm groups, although the fertility rates of eggs in both groups were decreased and the body weight gain of F(1) females in the E(2) 3-ppm group was significantly suppressed. In the histopathological examination of F(1) chicks maintained up to 10 weeks of age, persistent right oviduct and atrophy of the oviduct gland were observed in females of E(2)-treatment groups with significantly high incidences. Moreover, cystic dilatation and tubular degeneration of the seminiferous tubules and atrophy of the cloacal gland were also observed in males of the E(2)-treatment groups. Thus, the dietary treatment of low dose E(2) (even 0.3 ppm) to parent quails resulted in decreased viability and induction of abnormalities in the oviduct, testis and cloacal gland in F(1) chicks maintained up to 10 weeks of age. These results suggest that additional endpoints such as observations of F(1) chicks until 10 weeks of age, histopathology of F(1) chicks at 14 days and 10 weeks of age and blood VTG concentration in parent quails would be useful and sensitive endpoints for evaluating estrogenic endocrine disrupting effects in the avian reproduction study.