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The central nervous system (CNS) diseases are still a major cause of morbidity and mortality throughout the world, which imposes heavy burden on the development of society. Ethionine is a non-proteinogenic amino acid having similar chemical structure and activity to that of methionine, with which it competes. Previous studies have confirmed that ethionine affects various cellular functions by inhibiting the biosynthesis of proteins, RNA, DNA, and phospholipids, or all of them. The relationship of ethionine with some CNS diseases, including neural tube defects, has been investigated recently. However, the detailed effects of ethionine on the nerve cell bioactivities and the underlying mechanisms have not been fully explored. Herein, we systematically investigated the influences of ethionine on the proliferation, differentiation, and apoptosis of neural stem cells (NSCs) and post-mitotic nerve cells. We demonstrated that ethionine inhibited cell viability by disrupting the balance between proliferation and apoptosis, prevented NSCs from differentiating into neurons and astrocytes, and blocked cell progression from G1 to S phase via reducing cyclin D1 function in nerve cells including NSCs, a mouse hippocampal neuron cell line (HT-22), and a mouse brain neuroma cell line (Neuro-2a). We speculated that the inhibitory effect of ethionine on cell viability and differentiation are associated with increased reactive oxygen species production. Our results also supported the concept that ethionine may be an underlying cause of abnormal folate metabolism-induced CNS diseases. Our findings may provide important direction for the application of abnormal folate metabolism-induced CNS diseases in future NSC-based therapies., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The choline-deficient, ethionine-supplemented (CDE) dietary model induces chronic liver damage, and stimulates liver progenitor cell (LPC)-mediated repair. Long-term CDE administration leads to hepatocellular carcinoma in rodents and lineage-tracing studies show that LPCs differentiate into functional hepatocytes in this model. The CDE diet was first modified for mice by our laboratory by separately administering choline-deficient chow and ethionine in the drinking water (CD+E diet). Although this CD+E diet is widely used, concerns with variability in weight loss, morbidity, mortality and LPC response have been raised by researchers who have adopted this model. We propose that these inconsistencies are due to differential consumption of chow and ethionine in the drinking water, and that incorporating ethionine in the choline-deficient chow, and altering the strength, will achieve better outcomes. Therefore, C57Bl/6 mice, 5 and 6 weeks of age, were fed an all-inclusive CDE diet of various strengths (67% to 100%) for 3 weeks. The LPC response was quantitated and cell lines were derived. We found that animal survival, LPC response and liver damage are correlated with CDE diet strength. The 67% and 75% CDE diet administered to mice older than 5 weeks and greater than 18 g provides a consistent and acceptable level of animal welfare and induces a substantial LPC response, permitting their isolation and establishment of cell lines. This study shows that an all-inclusive CDE diet for mice reproducibly induces an LPC response conducive to in vivo studies and isolation, whilst minimizing morbidity and mortality., (© 2015. Published by The Company of Biologists Ltd.)

The roles of methionine metabolism in bovine preimplantation embryo development were investigated by using ethionine, an antimetabolite of methionine. In vitro produced bovine embryos that had developed to the 5-cell stage or more at 72 h after the commencement of in vitro fertilization (IVF) were then cultured until day 8 (IVF = day 0) in medium supplemented with 0 (control), 1, 5 and 10 mM ethionine. Compared with the blastocyst development in the control (40.0%), ethionine at 10 mM almost completely blocked blastocyst development (1.1%, P<0.001), and this concentration was used in the following experiments. Methionine added at the same concentration (10 mM, a concentration control of ethionine) did not cause such an intense developmental inhibition. Development to the compacted morula stage on day 6 was not affected by 10 mM ethionine treatment. S-adenosylmethionine (SAM) added to the ethionine treatment partly restored the blastocyst development. Semiquantitative reverse transcription-polymerase chain reaction analysis of cell lineage-related transcription factors in day 6 compacted morulae showed that the expressions of NANOG and TEAD4 were increased by ethionine treatment relative to the control (P<0.01). Furthermore, immunofluorescence analysis of 5-methylcytosine revealed that DNA was hypomethylated in the ethionine-treated day 6 morulae compared with the control (P<0.001). These results demonstrate that the disruption of methionine metabolism causes impairment of the morula-to-blastocyst transition during bovine preimplantation development in part via SAM deficiency, indicating the indispensable roles of methionine during this period. The disruption of methionine metabolism may cause hypomethylation of DNA and consequently lead to the altered expression of developmentally important genes, which then results in the impairment of blastocyst development.

Activation of myofibroblasts (MF) and extracellular matrix (ECM) deposition predispose the expansion and differentiation of liver progenitor cells (LPC) during chronic liver injury. Because Kupffer cells (KC) are active modulators of tissue response and fibrosis, we analyzed their role in a model of LPC proliferation. A choline-deficient diet, supplemented by ethionine (CDE) was administrated to C57Bl/6J mice that were depleted of KC by repeated injections of clodronate (CLO) and compared to PBS-injected mice. On CDE, massive KC activation was observed in the PBS group, but this was blunted in CLO-treated mice. The depletion of KC did not influence LPC proliferation but reduced their invasive behavior. Instead of being found far into the parenchyma, as was found in the PBS group (mean distance from portal vein: 209 μm), LPC of CLO mice remained closer to the portal area (138 μm), forming aggregates and phenotypically resembling cells of biliary lineage. Notably, removal of KC was also associated with a significant decrease in amount of MF and ECM and in the expression of profibrotic factors. Thus, besides ECM and MF, KC are also a significant component of the microenvironmental changes preceding LPC expansion. Depletion of KC may limit the LPC parenchymal invasion through a deficiency in chemoattracting factors, reduced activation of MF, and/or a paucity of the ECM framework necessary for cell motility., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Administration of ethionine to female rats caused a rapid and severe decline in liver ATP and inhibited hepatic protein synthesis in association with hypophosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 70-kDa ribosomal protein S6 kinase (S6K1), two key regulatory proteins involved in initiation of mRNA translation. Phosphorylation of both regulatory proteins is mediated through a signaling pathway that involves the mammalian target of rapamycin (mTOR). Recent studies indicate that AMP-activated protein kinase (AMPK) plays a role in the cellular response to environmental stresses, which deplete ATP, and suppresses protein synthesis through downregulated mTOR signaling. We investigated the possible involvement of AMPK in the ethionine-induced inhibition of protein synthesis. The administration of ethionine surprisingly decreased AMPK activity compared with controls despite ATP depletion. We conclude that inhibition of protein synthesis by ethionine is due to AMPK-independent inhibition of mTOR signaling following ATP depletion.

The goal of the present study was to measure the total glutathione level and glutathione reductase activity in bovine erythrocytes and liver biopsy. Five cows were injected intraperitoneally with DL-ethionine (12.5 mg/kg B.W.), and two control cows were injected with normal saline (0.9% NaCl). Ultrasonography guided liver biopsy, and blood samples were collected at 0, 4, 7 and 10 days after injection. The hepatic total glutathione level was significantly increased on Days 7 (p<0.05) and 10 (p<0.01), and hepatic glutathione reductase activity was significantly increased on Days 4 (p<0.05), 7 (p<0.01) and 10 (p<0.01). There were insignificant changes in the erythrocytic total glutathione level. The present study demonstrated that liver biopsy is a valuable tool for detecting oxidative stress and for diagnosing hepatic dysfunction in cattle from the viewpoint of the status of glutathione and glutathione reductase.

An efficient method for creating a DNA library is presented in which gene mutagenesis and recombination can be introduced by integrating error-prone PCR with a staggered extension process in one test tube. In this process, less than 15 cycles of error-prone PCR are used to introduce random mutations. After precipitated and washed with ethanol solution, the error-prone PCR product is directly used both as template and primers in the following staggered extension process to introduce DNA recombination. The method was validated by using adenosyl-methionine (AdoMet) synthetase gene, sam1, as a model. The full-length target DNA fragment was available after a single round. After being selected with a competitive inhibitor, ethionine, a mutated gene was obtained that increased AdoMet accumulation in vivo by 56%.

Bone marrow cells (BMCs) have been reported to behave as tissue-specific stem cells in some organs and to participate in tumorigenesis. However, the roles of BMCs in hepatic regeneration and carcinogenesis are still unknown. A choline-deficient, ethionine-supplemented (CDE) diet leads to the appearance of oval cells, a type of hepatic progenitor cell, and activates their replication. Furthermore, this type of diet induces preneoplastic nodules and hepatocellular carcinomas (HCCs) derived from oval cell progenitors. The aims of this study were to determine whether oval cells are derived from BMCs and whether preneoplastic nodules or HCCs originate from BMCs in the CDE diet rat model. To clarify the origin of constituent cells in the liver, we transplanted BMCs from green fluorescent protein (GFP) transgenic female rats into male Lewis rats, which were then exposed to a CDE diet to induce hepatocarcinogenesis. Some oval cells showed both donor-derived GFP expression and the recipient-specific Y chromosome, indicating that donor BMCs fused with recipient oval cells. Several preneoplastic nodules (precancerous lesions) identified by their glutathione S-transferase placental (GSTp) positivity were induced by CDE treatment. However, these preneoplastic GSTp-positive nodules were not GFP positive. In conclusion, this study has produced two major findings. First, BMCs fuse with some oval cells. Second, BMC-fused oval cells and BMCs might not have malignant potential in the CDE-treated rat model.

Background: S-Adenosylmethionine synthetase (AdoMetS) catalyzes the formation of S-Adenosylmethionine (AdoMet), the major methyl group donor in cells. AdoMet-mediated methylation of DNA is known to have regulatory effects on DNA transcription and chromosome structure. Transcription of environmental-responsive genes was demonstrated to be mediated via DNA methylation in dinoflagellates., Results: A full-length cDNA encoding AdoMetS was cloned from the dinoflagellate Crypthecodinium cohnii. Phylogenetic analysis suggests that the CcAdoMetS gene, is associated with the clade of higher plant orthrologues, and not to the clade of the animal orthrologues. Surprisingly, three extra stretches of residues (8 to 19 amino acids) were found on CcAdoMetS, when compared to other members of this usually conserved protein family. Modeled on the bacterial AdeMetS, two of the extra loops are located close to the methionine binding site. Despite this, the CcAdoMetS was able to rescue the corresponding mutant of budding yeast. Southern analysis, coupled with methylation-sensitive and insensitive enzyme digestion of C. cohnii genomic DNA, demonstrated that the AdoMetS gene is itself methylated. The increase in digestibility of methylation-sensitive enzymes on AdoMet synthetase gene observed following the addition of DNA methylation inhibitors L-ethionine and 5-azacytidine suggests the presence of cytosine methylation sites within CcAdoMetS gene. During the cell cycle, both the transcript and protein levels of CcAdoMetS peaked at the G1 phase. L-ethionine was able to delay the cell cycle at the entry of S phase. A cell cycle delay at the exit of G2/M phase was induced by 5-azacytidine., Conclusion: The present study demonstrates a major role of AdoMet-mediated DNA methylation in the regulation of cell proliferation and that the CcAdoMetS gene is itself methylated.

Birt-Hogg-Dube (BHD) is a tumor suppressor gene disorder characterized by skin hamartomas, cystic lung disease, and renal cell carcinoma. The fact that hamartomas, lung cysts, and renal cell carcinoma can also occur in tuberous sclerosis complex (TSC) suggests that the BHD and TSC proteins may function within a common pathway. To evaluate this hypothesis, we deleted the BHD homolog in Schizosaccharomyces pombe. Expression profiling revealed that six permease and transporter genes, known to be down-regulated in Deltatsc1 and Deltatsc2, were up-regulated in Deltabhd, and levels of specific intracellular amino acids known to be low in Deltatsc1 and Deltatsc2 were elevated in Deltabhd. This "opposite" profile was unexpected, given the overlapping clinical phenotypes. The TSC1/2 proteins inhibit Rheb in mammals, and Tsc1/Tsc2 inhibit Rhb1 in S. pombe. Expression of a hypomorphic allele of rhb1(+) dramatically increased permease expression levels in Deltabhd but not in wild-type yeast. Loss of Bhd sensitized yeast to rapamycin-induced increases in permease expression levels, and rapamycin induced lethality in Deltabhd yeast expressing the hypomorphic Rhb1 allele. In S. pombe, it is known that Rhb1 binds Tor2, and Tor2 inhibition leads to up-regulation of permeases including those that are regulated by Bhd. Our data, therefore, suggest that Bhd activates Tor2. If the mammalian BHD protein, folliculin, similarly activates mammalian target of rapamycin, it will be of great interest to determine how mammalian target of rapamycin inhibition in BHD patients and mammalian target of rapamycin activation in TSC patients lead to overlapping clinical phenotypes.

Keratin polypeptides 8 and 18 (K8/K18) are the major intermediate filament proteins of pancreatic acinar cells and hepatocytes. Pancreatic keratin function is unknown, whereas hepatocyte keratins protect from mechanical and non-mechanical forms of stress. We characterized steady-state pancreatic keratin expression in Balb/c mice after caerulein and choline-deficient ethionine-supplemented diet (CDD), or on exposure to the generalized stresses of heat and water immersion. Keratins were studied at the protein, RNA and organizational levels. Isolated acini were used to study the role of nuclear factor (NF)-kappaB using selective inhibitors. Keratins were found to be abundant proteins making up 0.2%, 0.3% and 0.5% of the total cellular protein of pancreas, liver and small intestine, respectively. Caerulein and CDD caused a threefold transcription-mediated overall increase in K8/K18/K19/K20 proteins. Keratin overexpression begins on tissue recovery, peaks 2 days after caerulein injection, or 1 day after CDD discontinuation, and returns to basal levels after 10 days. K19/K20-containing cytoplasmic filaments are nearly absent pre-injury but form post-injury then return to their original membrane-proximal distribution after 10 days. By contrast, generalized stresses of heat or water-immersion stress do not alter keratin expression levels. Caerulein-induced keratin overexpression is associated with NF-kappaB activation when tested using ex vivo acinar cell cultures. In conclusion, keratins are abundant proteins that can behave as stress proteins in response to tissue-specific but not generalized forms of injury. Pancreatic keratin overexpression is associated with NF-kappaB activation and may serve unique functions in acinar or ductal cell response to injury.

Oval cells, putative hepatic stem cells, could potentially provide a novel solution to the severe shortage of donor livers, because of their ability to proliferate and differentiate into functional hepatocytes. We have previously demonstrated that oval cells can be induced to differentiate into cells with morphologic, phenotypic, and functional characteristics of mature hepatocytes. In this study, we have established a new model combining ethionine treatment with partial hepatectomy to activate oval cells, then developed a procedure utilizing selective enzymatic digestion and density gradient centrifugation to isolate and purify such cells from heterogeneous liver cell population. We identified oval cells by their morphological characteristics and phenotypic properties, thereby providing definitive evidence of the presence of hepatic stem-like cells in adult rat livers. Viewed by transmission electron microscopy, they were small cells with ovoid nuclei, a high nucleus/cytoplasm ratio and few organelles, including mitochondria and endoplasmic reticulum. Flow cytometric assay showed that these cells highly expressed OV-6, cytokeratin-19 (CK-19) and albumin. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis displayed that the freshly isolated cells co-expressed albumin, cytokeratin-7 (CK-7) and CK-19 mRNA, indicating that they were essentially bipotential hepatic stem-like cells. Furthermore, we set up a culture system containing growth factors and a fibroblast feeder layer, to provide nourishment to these cells. Thus, we were able to culture them in vitro for more than 3 months, with the number of cells doubling 100 times. Gene expressions of albumin, CK-7 and CK-19 in the cells derived from the expanding colonies at day 95 were confirmed by RT-PCR analysis. These data suggested that the hepatic oval cells derived from adult rat livers possess a high potential to proliferate in vitro with a large increase in number, while maintaining the bipotential nature of hepatic stem cells.

IL-10 is produced by a large variety of cells including monocytes, macrophages, B and T lymphocytes, as well as natural killer cells and is an important suppressor for both immunoproliferative and inflammatory responses. IL-10 exerts antifibrotic effects in the liver, and decreased monocyte synthesis of IL-10 is well documented in alcoholic cirrhosis. Intracellular deficiency of S-adenosylmethionine (AdoMet) is a hallmark of toxin-induced liver injury. Although the administration of exogenous AdoMet attenuates this injury, the mechanisms of its actions are not fully established. This study was performed to investigate the effect of exogenous AdoMet on IL-10 production in LPS-stimulated RAW 264.7 cells, a murine macrophage cell line. Our results demonstrated that exogenous AdoMet administration enhanced both protein production and gene expression of IL-10 in RAW 264.7 cells. Ethionine, an inhibitor for methionine adenosyltransferases, inhibited LPS-stimulated IL-10 both at the protein and mRNA levels. Exogenous AdoMet increased the intracellular cAMP concentration as early as 3 h and continued for 24 h after AdoMet treatment; however, the inhibitors for both adenylyl cyclase and PKA did not significantly affect IL-10 production. On the basis of these results, we conclude that AdoMet administration may exert its anti-inflammatory and hepatoprotective effects, at least in part, by enhancing LPS-stimulated IL-10 production.

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the R. prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a K(T) of 2.3 micro M. Transport was inhibited by S-adenosylethionine and S-adenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of R. prowazekii and in Rickettsia typhi. By screening Escherichia coli clone banks for AdoMet transport, the R. prowazekii gene coding for a transporter, RP076 (sam), was identified. AdoMet transport in E. coli containing the R. prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.

Background & Aims: Macrophage migration inhibitory factor (MIF), originally described as an inhibitor of the random migration of macrophages, has been shown recently to be involved in the pathogenesis of several inflammatory diseases such as sepsis. The aim of this study was to clarify the role of MIF in acute pancreatitis (AP)., Methods: Hemorrhagic necrotizing pancreatitis and edematous pancreatitis were induced by the injection of taurocholic acid (TCA pancreatitis) and cerulein (cerulein pancreatitis), respectively, on male Wistar rats. MIF levels in ascitic fluids, serum, and the organs were determined. The effects of anti-MIF antibody were examined on the prognosis of rats with TCA pancreatitis and of female CD-1 mice with choline-deficient, ethionine-supplemented, diet-induced model of severe AP. In addition, serum MIF levels in AP patients and in healthy controls were measured., Results: Serum and ascitic MIF levels in TCA pancreatitis were increased rapidly and decreased gradually thereafter. Ascitic MIF levels were also increased in cerulein pancreatitis, but to a lesser degree. MIF level was increased in the lung in TCA pancreatitis, but not in the pancreas and the liver. Prophylactic (1 hour before and immediately after induction) administration of anti-MIF antibody significantly improved the survival rate of rats with TCA pancreatitis. The survival rate of mice with severe AP was also improved significantly by the antibody treatment. Serum MIF levels were higher in severe AP patients than mild AP patients and healthy controls., Conclusions: These results suggest a role of MIF in the pathogenesis of severe AP.

To examine the effect of ethanol on hepatocarcinogenesis induced by a choline-deficient, ethionine-supplemented (CDE) diet, rats were fed either an ethanol-supplemented diet or ethanol-free, isocaloric diet for 2 months, followed by a CDE diet or control diet for up to 8 months. Changes to cellular composition and pattern of gene expression in the liver were determined at 0 and 3 days, and 1, 2 and 3 weeks after commencing the CDE diet, using histological/immunochemical techniques and northern analysis. Oval cells in the liver were identified morphologically and by expression of pi-glutathione S-transferase (pi-GST), alpha-fetoprotein (AFP) and the embryonic isoform of pyruvate kinase (M2-PK). Oval cell numbers and changes in the pattern of gene expression induced by the CDE diet were accelerated by pre-treatment with ethanol. At all stages, the proportion of oval cells in the test group exceeded that in controls. After 1 week, oval cells had spread sufficiently from the periportal region to be observed pericentrally in test animals and by 3 weeks, extensive formation of ductal structures was apparent, which were absent in controls. Additionally, M2-PK and AFP mRNA were detected earlier, and in greater abundance in animals pre-treated with ethanol. After 8 months of CDE treatment, one or two small hepatic foci (<10 hepatocytes), strongly positive for pi-GST, were detected in the liver of ethanol-pre-treated animals. These foci were absent in CDE-treated animals; however, animals pre-treated with ethanol followed by chronic CDE treatment showed increased size (>40 hepatocytes) and numbers of foci, correlating with the extent of liver damage and varying from 5 to 50% of the liver section. Our data suggest that ethanol pre-treatment potentiates the short-term effects of the CDE diet by enhancing oval cell proliferation, while chronic CDE administration enhances the appearance of pre-malignant hepatic foci that are observed with ethanol pre-treatment alone.

Cystathionine gamma-synthase (CGS) catalyzes the first committed step of Met biosynthesis in plants. We have previously shown that expression of the gene for CGS is feedback-regulated at the level of mRNA stability, and that the amino acid sequence encoded by the first exon of the CGS gene itself is responsible for the regulation (Chiba, Y., Ishikawa, M., Kijima, F., Tyson, R. H., Kim, J., Yamamoto, A., Nambara, E., Leustek, T., Wallsgrove, R. M., and Naito, S. (1999) Science 286, 1371-1374). To identify the functional region within CGS exon 1, deletion analysis was performed. The results showed that the 41-amino acid region of exon 1 highly conserved among plants is necessary and sufficient for the regulation. Analyses of in vivo and in vitro generated mutations that abolish the regulation identified the functionally important amino acids as 11-13 residues within this conserved region. The importance of these residues was confirmed by deletion analysis within the conserved region. These studies identified the functional region of CGS exon 1 required for the posttranscriptional autoregulation of the CGS gene as (A)RRNCSNIGVAQ(I), with uncertainty of the first and last residues. This sequence is almost perfectly conserved among CGS sequences of higher plants but cannot be found elsewhere in the public databases.

Met-overaccumulating mutants provide a powerful genetic tool for examining both the regulation of the Met biosynthetic pathway and in vivo developmental responses of gene expression to altered Met levels. We have previously reported the identification of two Arabidopsis thaliana Met over-accumulation (mto) mutants, mto1-1 and mto2-1, that carry mutations in the genes encoding cystathionine gamma-synthase (CGS) and threonine synthase (TS), respectively. A third mutant, mto3-1, has recently been reported to carry a mutation in the gene encoding S-adenosylmethionine synthetase 3 (SAMS3). Here, we report the isolation of a new ethionine-resistant A. thaliana mutant that over-accumulates soluble Met approximately 20-fold in young rosettes. The causal mutation was determined to be a single, recessive mutation that was mapped to chromosome 3. Sequence analysis identified a single nucleotide change in the gene encoding SAMS3 that was distinct from the mto3-1 mutation and altered the amino acid sequence of the enzyme active site. This mutation was therefore referred to as mto3-2. Although Met over-accumulation in the mto3-2 mutant was similar to that in the mto2-1 mutant, CGS mRNA levels did not respond to the mto3-2 mutation and were similar to that in equivalent wild-type plants.

Oval cells are bipotential liver stem cells able to differentiate into hepatocytes and bile duct epithelia. In normal adult liver oval cells are quiescent, existing in low numbers around the periportal region, and proliferate following severe, prolonged liver trauma. There is evidence implicating oval cells in the development of hepatocellular carcinoma, and hence the availability of an immortalized oval cell line would be invaluable for the study of liver cell lineage differentiation and carcinogenesis. A novel approach in the generation of cell lines is the use of the p53 knockout mouse. Absence of p53 allows a cell to cycle past the normal Hayflick limit, rendering it immortalized, although subsequent genetic alterations are thought necessary for transformation. p53 knockout mice were fed a choline-deficient, ethionine-supplemented diet, previously shown to increase oval cell numbers in wild-type mice. The oval cells were isolated by centrifugal elutriation and maintained in culture. Colonies of hepatic cells were isolated and characterized with respect to phenotype, growth characteristics and tumorigenicity. Analysis of gene expression by Northern blotting and immunocytochemistry suggests they are oval-like cells by virtue of albumin and transferrin expression, as well as the oval cell markers alpha fetoprotein, M(2)-pyruvate kinase and A6. Injection into athymic nude mice shows the cell lines are capable of forming tumors which phenotypically resemble hepatocellular carcinoma. Thus, the use of p53 null hepatic cells successfully generated immortalized and tumorigenic hepatic stem cell lines. The results presented support the idea that deleting p53 allows immortalization and contributes to the transformation of the oval-like cell lines. Further, the tumorigenic status of the cell lines is direct evidence for the participation of oval cells in the formation of hepatocellular carcinoma.

The committing step in Met and S-adenosyl-L-Met (SAM) synthesis is catalyzed by cystathionine gamma-synthase (CGS). Transgenic Arabidopsis plants overexpressing CGS under control of the cauliflower mosaic virus 35S promoter show increased soluble Met and its metabolite S-methyl-Met, but only at specific stages of development. The highest level of Met and S-methyl-Met was observed in seedling tissues and in flowers, siliques, and roots of mature plants where they accumulate 8- to 20-fold above wild type, whereas the level in mature leaves and other tissues is no greater than wild type. CGS-overexpressing seedlings are resistant to ethionine, a toxic Met analog. With these properties the transgenic lines resemble mto1, an Arabidopsis, CGS-mutant inactivated in the autogenous control mechanism for Met-dependent down-regulation of CGS expression. However, wild-type CGS was overexpressed in the transgenic plants, indicating that autogenous control can be overcome by increasing the level of CGS mRNA through transcriptional control. Several of the transgenic lines show silencing of CGS resulting in deformed plants with a reduced capacity for reproductive growth. Exogenous feeding of Met to the most severely affected plants partially restores their growth. Similar morphological deformities are observed in plants cosuppressed for SAM synthetase, even though such plants accumulate 250-fold more soluble Met than wild type and they overexpress CGS. The results suggest that the abnormalities associated with CGS and SAM synthetase silencing are due in part to a reduced ability to produce SAM and that SAM may be a regulator of CGS expression.

Hepatic stem cells (oval cells) proliferate within the liver after exposure to a variety of hepatic carcinogens and can generate both hepatocytes and bile duct cells. Oval cell proliferation is commonly seen in the preneoplastic stages of liver carcinogenesis, often accompanied by an inflammatory response. Tumor necrosis factor (TNF), an inflammatory cytokine, is also important in liver regeneration and hepatocellular growth. The experiments reported here explore the relationship among the TNF inflammatory pathway, liver stem cell activation, and tumorigenesis. We demonstrate that TNF is upregulated during oval cell proliferation induced by a choline-deficient, ethionine-supplemented diet and that it is expressed by oval cells. In TNF receptor type 1 knockout mice, oval cell proliferation is substantially impaired and tumorigenesis is reduced. Oval cell proliferation is impaired to a lesser extent in interleukin 6 knockout mice and is unchanged in TNF receptor type 2 knockout mice. These findings demonstrate that TNF signaling participates in the proliferation of oval cells during the preneoplastic phase of liver carcinogenesis and that loss of signaling through the TNF receptor type 1 reduces the incidence of tumor formation. The TNF inflammatory pathway may be a target for therapeutic intervention during the early stages of liver carcinogenesis.

Background & Aims: Intercellular adhesion molecule 1 (ICAM-1) and neutrophils play important roles in many inflammatory processes, but their importance in both acute pancreatitis and pancreatitis-associated lung injury has not been defined., Methods: To address this issue, mice that do not express ICAM-1 were used and depleted of neutrophils by administration of antineutrophil serum. Pancreatitis was induced by administering either supramaximal doses of the secretagogue cerulein or feeding a choline-deficient, ethionine-supplemented diet. The severity of pancreatitis was evaluated by quantitating serum amylase, pancreatic edema, acinar cell necrosis, and pancreas myeloperoxidase activity (i.e., neutrophil content). Lung injury was evaluated by quantitating lung myeloperoxidase activity and pulmonary microvascular permeability. ICAM-1 was quantitated by enzyme-linked immunosorbent assay and was localized by light-microscopic immunohistochemistry., Results: It was found that serum, pancreas, and lung ICAM-1 levels increase during pancreatitis. Both pancreatitis and the associated lung injury are blunted, but not completely prevented, in mice deficient in ICAM-1. Neutrophil depletion also reduces the severity of both pancreatitis and lung injury. However, the combination of neutrophil depletion with ICAM-1 deficiency does not reduce the severity of pancreatitis or lung injury to a greater extent than either neutrophil depletion or ICAM-1 deficiency alone. Neither pancreatitis nor pancreatitis-associated lung injury are completely prevented by ICAM-1 deficiency, neutrophil depletion, or combined ICAM-1 deficiency plus neutrophil depletion., Conclusions: The observations indicate that ICAM-1 plays an important, neutrophil-mediated, proinflammatory role in pancreatitis and pancreatitis-associated lung injury. The studies also indicate that ICAM-1 and neutrophil-independent events also contribute to the evolution of pancreatitis and lung injury in these models.

The present study was designed to define whether maximal removal rate of indocyanine green (ICG Rmax), plasma cyclic 3',5'-adenosine monophosphate (cAMP) response to exogenous glucagon (peak to basal ratio of cAMP level: P/B cAMP) and plasma half-life of galactose (t1/2 galactose) can measure the hepatic functional reserve of fatty liver prepared in rats fed choline-deficient (9 weeks), 2% cholesterol (2 weeks) or 0.25% DL-ethionine (2 weeks) diet. Although changes in cholesterol and phospholipid values in serum during feeding periods differed among the models, histopathologic examinations in the liver of almost all animals revealed intermediate to severe fatty liver with or without fibrosis at each termination. ICG Rmax and P/B cAMP were significantly decreased in rats fed choline-deficient or DL-ethionine diet, implying reductions in hepatic functional mass and disturbances in hepatic cAMP production. Meanwhile, t1/2 galactose showed no change in any of the models, suggesting that glucose metabolisms in the models used may be preserved. These findings demonstrate that ICG Rmax and P/B cAMP can apply to measurement of hepatic surviving reserve of fatty liver with fibrosis.

Variations in the genomic DNA methylation level have been shown to be an epigenetic inheritable modification affecting, among other targets, the sister chromatid exchange (SCE) rate in mammalian cells in vitro. The inheritable increase in SCE rate in affected cell populations appears as a puzzling phenomenon in view of the well established relation between SCE and both mutagenesis and carcinogenesis. In the present work we demonstrate that, in a treated cell population, demethylation could be responsible for the inheritable induction of damage proneness affecting both damage induction and repair. Normal and ethionine or azacytidine treated Chinese hamster ovary cells, subclone K1 (CHO-K1), were challenged with UV light (UV) or mitomycin-C (MMC) at different times from the demethylating treatment. The SCE rate was measured with two main objects in view: (i) the induction of synergism or additivity in combined treatments, where mutagen (UV or MMC) pulse is supplied from 0 to 48 h after the end of the demethylating treatment; and (ii) the pattern of damage extinction, for the duration of up to six cell cycles after the end of the combined (demethylating agent + mutagen) treatment. Results indicate both a synergism in SCE induction by mutagens in demethylated cells even if supplied up to four cell cycles after the end of the demethylation treatment and a delay in recovery of induced damage, compared with normally methylated cells. These data are discussed in the light of the supposed mechanism of SCE increase and of the possible biological significance in terms of mutagenesis and carcinogenesis.

A Candida albicans oligopeptide transport gene, OPT1, was cloned from a C. albicans genomic library through heterologous expression in the Saccharomyces cerevisiae di-/tripeptide transport mutant PB1X-9B. When transformed with a plasmid harbouring OPT1, S. cerevisiae PB1X-9B, which did not express tetra-/pentapeptide transport activity under the conditions used, was conferred with an oligopeptide transport phenotype, as indicated by growth on the tetrapeptide Lys-Leu-Leu-Gly, sensitivity to toxic tetra- and pentapeptides, and an increase in the initial uptake rate of the radiolabelled tetrapeptide Lys-Leu-Gly-[3H]Leu. The level of oligopeptide transport was found to be influenced in the heterologous host by the source of nitrogen used for growth. The entire 3.8 kb fragment containing the oligopeptide transport activity was sequenced and an ORF of 2349 nucleotides containing a 58 nucleotide intron was identified. The deduced protein product of 783 amino acid residues contained 12 hydrophobic regions suggestive of a membrane transport protein. Sequence comparisons revealed that similar proteins are encoded by genes from S. cerevisiae and Schizosaccharomyces pombe and that OPT1 is not a member of the ABC or PTR membrane transport families.

Previous studies showed that high concentrations of methionine (> 1 mM) inhibited aggregation and fruiting body formation in Myxococcus xanthus (E. Rosenberg, D. Filer, D. Zafriti, and S. H. Kindler, J. Bacteriol. 115: 29-34, 1973, and J. M. Campos and D. R. Zusman, Proc. Natl. Acad. Sci. USA 72:518-522, 1975). However, the mechanism for the inhibition was unclear. In this study, we found that high levels of methionine inhibited the biosynthesis of S-adenosylmethionine (SAM) and that reduced intracellular levels of SAM are correlated with defective chemotactic movements and reduced developmental gene expression. In addition, we found that methionine analogs and high concentrations of amino acids which are known to affect SAM synthesis in other bacteria, such as threonine, lysine, and isoleucine, also caused reduced cellular levels of SAM and blocked fruiting body formation in M. xanthus. These results indicate that SAM is required for development of M. xanthus and the inhibitory effect of methionine on development results, at least in part, from its blocking of the biosynthesis of SAM.

An electron microscopic, immunocytochemical, and enzyme cytochemical analysis of the previously established oval cell lines OC/CDE 6 and OC/CDE 22 was performed to characterize the phenotype and differentiation patterns of long-term cultures of oval cells. It was found that alpha-fetoprotein, albumin, and cytokeratin 19 are present in all cultured cells. This indicates that oval cells constitute a population of immature cells expressing features of the antigenic phenotype of both the hepatocyte and bile ductular cell lineages. An electron microscopic examination revealed a gradual alteration in the ultrastructure of oval cells toward hepatocyte-like cells. The majority of the oval cells were positive for glucose-6-phosphatase activity. A particularly striking observation was that oval cells were heterogeneous in terms of peroxisome content. Only about 50% of the oval cells had peroxisomes in the cytoplasm, these cells probably being part of the hepatocyte lineage. The other cultured cells did not reveal catalase activity and probably represented cells committed to the bile ductular cell lineage. An addition of clofibrate to the culture medium resulted in a marked peroxisome proliferation in all oval cells, indicating that oval cells might be able to change their differentiation pathway depending on environmental influence toward the hepatocyte lineage. It is most intriguing that in oval cells with abundant cytoplasm peroxisome proliferation was accompanied by proliferation of the smooth endoplasmic reticulum (this is a morphological marker of mature hepatocytes). Taken together, our findings suggest that within the oval cell lines OC/CDE 6 and OC/CDE 22 cells undergoing a morphological and functional differentiation along the hepatocyte and bile ductular cell lineages are present.

Higher plant DNA is methylated at CG and CNG targets. In this study we have investigated the tobacco methylation system in tissue culture using the methylation inhibitors 5-azacytidine (5-azaC), dihydroxypropyladenine (DHPA) and ethionine (Ethi), and methylation-sensitive restriction endonucleases HpaII, MspI, HhaI, EcoRII, ScrFI, and Fnu4HI. Surprisingly, CAG/CTG sequences, contrary to CG doublets and CCG/CGG triplets, appeared to be refractory to the inhibitory effect of 5-azaC. Thus 5-azaC cannot be considered a general inhibitor of DNA methylation in tobacco cells. On the other hand, DHPA, the inhibitor of S-adenosylhomocysteine (SAH) hydrolase, and Ethi caused hypomethylation of both CAG/CTG and CCG/CGG triplets but not of the CG doublets. The sensitivity of triplet-specific methylation to the inhibition of SAH hydrolase suggests the possibility that plant-specific DNA methylation at CNG targets might be modulated by alterations of the SAH/S-Adenosylmethionine ratio in plant cells.

4-Aminophenylboronic acid (APBA), a known inhibitor of sporulation in Bacilli, as well as L-ethionine, a known inhibitor of chemotaxis in Enterobacteria, inhibited both sporulation and chemotactic behavior but not growth of Bacillus sphaericus. Both compounds also inhibited the methyl group turnover on the methyl-accepting chemotaxis protein (P53) in this microorganism. Sporulation of B. sphaericus was inhibited only when APBA was added to the growing culture before the late logarithmic stage. It was previously demonstrated that the ability of B. sphaericus to respond to chemoattractants sharply declines at the same age of the culture. Thus, it seems plausible that the action of both inhibitors upon sporulation may be attributed to the inhibition of some regulatory pathway common to chemotaxis and sporulation and involving protein methylation. Possible exchange of the nutrient depletion-related sensory information between chemotaxis and sporulation systems at the level of methyl group transfer is discussed.

A UV irradiation-induced mutant of B. sphaericus 2362 whose sporulation was inhibited neither by natural amino acids nor by L-ethionine was selected. The mutant (A61) grew slowly in rich amino acid medium and contained increased concentrations of heat-resistant spores throughout the growth. Slow growth of A61 was related to continuous presence of aging and sporulating cells even when the medium was rich in nutrients. The ability of the mutant to sense nutrient presence in the environment and to relate this information to systems regulating the switch from vegetative growth to sporulation seem to be damaged. A61 also demonstrated impaired chemotaxis. In contrast to the parent strain, only few amino acids elicited chemotactic response in A61. Methylation of the A61 methyl-accepting chemotaxis protein(s) was lower than that of the parent strain by one order of magnitude. Spontaneous fast-growing phenotypic revertants of A61 displayed sporulation behavior characteristic of B. sphaericus 2362. Their chemotaxis to amino acids was considerably improved. To some amino acids, it proved to be even stronger than in the original strain, B. sphaericus 2362. It is suggested, that methyl transfer events originating in the chemotactic system are involved in the triggering of sporulation, the A61 mutation being located in this signalling pathway.

We previously reported that a recombinant strain, SB412(pLGM304), was constructed from acidomycin-resistant mutants of Serratia marcescens and produced 200 mg of d-biotin per liter of a medium containing sucrose and urea (Sakurai et al., 1993a, b). In the present work, we intended to improve the d-biotin production. Both ethionine and S-2-aminoethylcysteine resistances were added to the host strain SB412, producing d-biotin at 20 mg l-1, and a resultant strain, ETA23, producing it at 33 mg l-1, was obtained. Cells of ETA23 did not maintain pLGM304 stably after greater than 30 generations under non-selective culture conditions. A new recombinant plasmid, pLGM304P, was constructed so as to be composed of pLGM304 and the parB locus, a plasmid-stabilizing element. ETA23 stably maintained pLGM304P after 50 generations under non-selective culture conditions. ETA23(pLGM304) produced 250 mg l-1 of d-biotin in a shaking flask under batch culture conditions and 500 mg l-1 in a jar fermentor under fed-batch culture conditions.

A UV irradiation-induced mutant of Bacillus sphaericus 2362 whose sporulation was inhibited neither by natural amino acids nor by L-ethionine was selected. The mutant (A61) grew slowly in rich amino acid medium and contained increased concentrations of heat-resistant spores throughout the growth. Slow growth of A61 was related to continuous presence of aging and sporulating cells even when the medium was rich in nutrients. Ability of the mutant to sense nutrient presence in the environment and to relate this information to systems regulating the switch from vegetative growth to sporulation seem to be damaged. A61 also demonstrated impaired chemotaxis. In contrast to the parent strain, only few amino acids elicited chemotactic response in A61. Methylation of the A61 methyl-accepting chemotaxis protein(s) was lower than that of the parent strain by one order of magnitude. Spontaneous fast-growing phenotypic revertants of A61 displayed sporulation behavior characteristic of B. sphaericus 2362. Their chemotaxis to amino acids was considerably improved. To some amino acids, it proved to be even stronger than in the original strain, B. sphaericus 2362. It is suggested, that methyl transfer events originating in the chemotactic system are involved in the triggering of sporulation, the A61 mutation being located in this signalling pathway.

4-Aminophenylboronic acid (APBA), a known inhibitor of sporulation in Bacilli, as well as L-ethionine, a known inhibitor of chemotaxis in Enterobacteria, inhibited both sporulation and chemotactic behavior but not growth of Bacillus sphaericus. Both compounds also inhibited the methyl group turnover on the methyl-accepting chemotaxis protein (P53) in this microorganism. Sporulation of B. sphaericus was inhibited only when APBA was added to the growing culture before the late logarithmic stage. It was previously demonstrated that the ability of B. sphaericus to respond to chemoattractants sharply declines at the same age of the culture. Thus, it seems plausible that the action of both inhibitors upon sporulation may be attributed to the inhibition of some regulatory pathway common to chemotaxis and sporulation and involving protein methylation. Possible exchange of the nutrient depletion-related sensory information between chemotaxis and sporulation systems at the level of methyl group transfer is discussed.

In a recent hypothesis [Selhub and Miller (1992) Am. J. Clin. Nutr. 55, 131-138], we proposed that homocysteinaemia arises from an interruption in S-adenosylmethionine's (AdoMet) coordinate regulation of homocysteine metabolism. The present study was undertaken to test a prediction of this hypothesis, that homocysteinaemia due to folate deficiency results from impaired homocysteine remethylation due to the deficiency and impaired synthesis of AdoMet, with the consequent inability of this metabolite to function as an activator of homocysteine catabolism through cystathionine synthesis. Rats were made folate-deficient by feeding them with a folate-free amino-acid-defined diet supplemented with succinylsulphathiazole. After 4 weeks, the deficient rats exhibited a 9.8-fold higher mean plasma homocysteine concentration and a 3.2-fold lower mean hepatic AdoMet concentration compared with folate-replete controls. Subsequent supplementation for 3 weeks of the folate-deficient rats with increasing levels of folate in the diet resulted in graded decreases in plasma homocysteine levels, accompanied by graded increases in hepatic AdoMet levels. Thus plasma homocysteine and hepatic AdoMet concentrations were inversely correlated as folate status was modified. In a second experiment, the elevation of plasma homocysteine in the deficient rats was found to be reversible within 3 days by intraperitoneal injections of ethionine. This effect of ethionine is thought to be exerted through S-adenosylethionine, which is formed in the liver of these rats. Like AdoMet, S-adenosylethionine is an activator of cystathionine beta-synthase and will effectively promote the catabolism of homocysteine through cystathionine synthesis. In crude liver homogenates of the rats treated with ethionine, cystathionine beta-synthase activity was 3-fold higher than that measured in homogenates from vehicle-treated controls.

In order to analyze the detoxication functions in rats with diabetes induced by streptozotocin, the authors administered to the diabetic animals two drugs, ethionine and benzo(a)pyrene, which affect mainly the liver and are metabolized through a glutathione conjugation process, and examined the changes in the content of glutathione and activities of related enzymes in the liver. In the liver of the rats with streptozotocin-induced diabetes, the total glutathione content, glutathione S-transferase activity and glutathione-insulin transhydrogenase activity were lower than those of normal rat livers, while the glutathione peroxidase activity showed high values. Although specific changes in the glutathione-related detoxication functions were observed in the rats to which ethionine or benzo(a)pyrene had been administered, these changes were not revealed under diabetic conditions. It is suggested that in diabetic rats responses to toxic stimuli are suppressed.

In order to assess the association of cow serum haptoglobin with fatty liver, haptoglobin concentrations were evaluated by single radial immunodiffusion in sera from cows after administration of ethionine (an analogue of methionine). Haptoglobin concentrations were found to be increased in the sera of the ethionine-administered cows. This result suggests that the induction of haptoglobin is associated in the pathogenesis of fatty liver.

Changes in chromatin structure of the HRS60 family of repetitive sequences in tobacco DNA were studied after hypomethylation induced with 5-azacytidine or DL-ethionine. The TaqI site in the HRS60 units lies in nucleosomal core regions and its cleavage is enhanced in the hypomethylated chromatin. In contrast, the cleavage of the Sau3AI site located in linker DNA does not depend on the level of methylation of DNA.

A variety of receptors on pancreatic acinar and duct cells regulate both pancreatic exocrine secretion and intracellular processes. These receptors are potential sites of action for therapeutic agents in the treatment of pancreatitis. Cholecystokinin (CCK) receptor antagonists, which may reduce the level of metabolic "stress" on acinar cells, have been shown to mitigate the severity of acute pancreatitis in a number of models. Not all studies have shown a benefit, however, and differences may exist between different structural classes of antagonists. Because increased pancreatic stimulation due to loss of feedback inhibition of CCK has been proposed to contribute to the pain of some patients with chronic pancreatitis, CCK receptor antagonists could also be of benefit in this setting. Somatostatin and its analogs diminish pancreatic secretion of water and electrolytes and have been effective in treating pancreatic fistulas and pseudocysts. These agents are also being evaluated for their ability to reduce pain in chronic pancreatitis (perhaps by reducing ductal pressure by diminishing secretory volume) and mitigating the severity of acute pancreatitis (possibly by reducing the metabolic load on acinar cells). Recently described secretin receptor antagonists may also have therapeutic value as a means of selectively inhibiting pancreatic secretion of water and electrolytes.

Plant DNA is distinguished from the DNA of all other organisms by its high content of 5-methylcytosine (5mC). 5mC levels may amount to 30% of total cytosines, distributed between the sequences CG and CXG. The results presented here show that the methylation status of CXG sequences could be influenced by culturing tobacco tissues on subtoxic concentrations of ethionine. The hypomethylating effect of ethionine, evaluated as the capability of MspI or HpaII to cleave the DNA, proved to be rather specific for CCG and differed from that of 5-azacytidine which did not discriminate between CG and CXG sequences.

As a first step toward developing the genetic potential of extremely thermophilic archaebacteria, mutant strains of Sulfolobus acidocaldarius were selected by plating cells directly on solid medium containing one of several growth inhibitors. Three spontaneous resistance phenotypes were observed (5-fluorouracil resistance, novobiocin resistance, and L-ethionine resistance), each at a different average frequency. Characterization of representative strains showed each of the three mutant phenotypes to provide a potentially useful genetic marker.

Control of methionine biosynthesis in Escherichia coli K12 was reinvestigated by using methionine-analogue-resistant mutants. Norleucine (NL) and alpha-methylmethionine (MM) were found to inhibit methionine biosynthesis directly whereas ethionine (Et) competitively inhibited methionine utilization. Adenosylation of Et to generate S-adenosylethionine (AdoEt) by cell-free enzyme from E. coli K12 was demonstrated. Tolerance of increasing concentrations of NL by E. coli K12 mutants is expressed serially as phenotypes NLR, NLREtR, NLRMMR and finally NLREtRMMR. All spontaneous NLR mutants had a metK mutation, whereas NTG-induced mutants had mutations in both the metK and metJ genes. The kinetics of methionine adenosylation by the E. coli K12 cell-free enzyme were found to be similar to those reported for the yeast enzyme, showing the typical lag phase at low methionine concentration and disappearance of this phase when AdoMet was included in the incubation mixture. NL extended the lag phase, and lowered the rate of subsequent methionine adenosylation, but did not affect the shortening of the lag phase of adenosylation by AdoMet.

The hypothesis that liver carcinogenesis may have as an important facet the early selection of carcinogen-resistant cells was tested in animals in which putative premalignant hepatocyte populations, hyperplastic nodules, were induced by 2-acetylaminofluorene or by ethionine. Hyperplastic nodules were observed to be resistant to the acute necrogenic effects of 2 hepatotoxins, CCl4 and dimethylnitrosamine, under conditions in which liver cell necrosis occurred in the liver surrounding the nodules. In addition, although [methyl-3H]dimethylnitrosamine was taken up to an equal degree in nodules and normal liver, the interactions with DNA, RNA, and protein in hyperplastic nodules were found to be about 50% less than in control liver. Hyperplastic nodules showed a marked decrease in uptake of [9-14C]-2-acetylaminofluorene, a finding that could account for the large decrease in labeling of DNA, RNA, and protein by [9-14C]-2acetylaminofluorene observed in the nodules. The results are consistent with and support the hypothesis that new hepatocyte populations that appear prior to cancer, during liver carcinogenesis, have as an important biological property a resistance to the cytotoxic effect of hepatocarcinogens. The basis for this resistance might be a decrease in uptake and/or a reduction in the level of activation of carcinogens.

Ethionine administered intraperitoneally to rats suffering from turpentine-induced inflammation preferentially reduced incorporation of 14C-leucine into fibrinogen, haptoglobin and other acute-phase proteins. The inhibitory effect was observed both in vivo and in liver slices obtained from ethionine-treated donors, while addition of ethionine to liver slices in vitro led to general reduction of synthesis of all liver and plasma proteins, including albumin. For comparison, the effects of galactosamine and actinomycin D on plasma protein synthesis in injured rats were also examined. It has been concluded that ethionine acts in the early phases of the acute-phase response, probably by inhibition of trauma-induced transcription of liver mRNA specific for acute-phase proteins.

Methionine analogs such as ethionine, selenomethionine, and trifluoromethionine all arrest growth and division of the yeast Saccharomyces cerevisiae. One analog, ethionine, caused cells of the yeast to arrest specifically within G1; reciprocal shift experiments showed that ethionine and alpha-factor arrested cells at the same step ("start"). The major effect of ethionine on synthesis of macromolecules was to reduce both the rate of appearance of 35S ribosomal precursor RNA and the rate of production of mature rRNA. Synthesis of protein was relatively unaffected by ethionine. Selenomethionine and trifluoromethionine caused cells to arrest randomly in the cell division cycle. Although treatment of cells with either selenomethionine or trifluoromethionine also reduced the rate of total RNA synthesis, each of these analogs had other effects that presumably prohibited completion of the cell cycle. We propose that the rate of rRNA production is an important regulatory event in the cell cycle.

The O2 dependence of cytochrome oxidase, cellular ATP/ADP ratio, and drug sulfation and glucuronidation were studied in isolated hepatocytes to examine secondary biochemical changes of hypoxia related to decreased cytochrome oxidase function. A decrease in cytochrome oxidase function as well as in cellular ATP/ADP ratio occurred at low O2 concentrations and their O2 dependencies gave p50 values of 3.2 and 2.8 microM O2, respectively. Changes in the ATP/ADP ratio corresponded directly with changes in the oxidation-reduction state of cytochrome oxidase. The p50 value for sulfation of acetaminophen (2.5 microM O2) corresponded with the values for ATP/ADP ratio and cytochrome oxidase. Addition of compounds that lowered cellular ATP/ADP ratio caused a corresponding inhibition of sulfation. Selective use of ethionine, an adenosyl-trapping agent, demonstrated that the O2 dependence of sulfation related directly to a decrease in the absolute ATP concentration. The p50 value for glucuronidation (5.2 microM O2) was higher than that for sulfation, but in spite of this difference, glucuronidation also correlated well with cellular ATP/ADP ratio when this ratio was decreased by either O2 limitation or metabolic inhibitors. These results demonstrate that the predominant limitation of drug sulfation and glucuronidation during hypoxia is due to decreased cytochrome oxidase function.