Application of a color-shift model with heterogeneous growth to a rat hepatocarcinogenesis experiment

Several hypotheses are established to describe the formation and progression of foci of altered hepatocytes (FAH). A common model of hepatocarcinogenesis is the mutation model (MM), which is based on the assumption that cells have to undergo multiple successive changes on their way from the normal to the malignant stage. This model describes growth and phenotype change of foci on the cellular level and is based on the assumption that single cells change their phenotype through mutation into the next stage and proliferate according to a linear stochastic birth-death process. In contrast, the color-shift model (CSM) was introduced by Kopp-Schneider et al. to describe that whole colonies of altered hepatocytes simultaneously alter their phenotype. In this paper two modifications of the color-shift model are considered which allow the growth rate to vary from focus to focus. All four models are compared with respect to their ability to predict number and radii of foci in a rat hepatocarcinogenesis experiment, in which rats were treated with the carcinogens N-nitrosomorpholine, 2-acetylaminofluoren and Phenobarbital. Maximum likelihood parameter estimates are given, and predicted and empirical FAH size distributions are visualized. The Cramer-von-Mises distance is used as a measure for the discrepancy between empirical and theoretical size distributions.     

Incorporating phenotype-dependent growth rates into the color-shift model for preneoplastic hepatocellular lesions

Multi-stage models occupy a central position in modeling the carcinogenesis process. These models formalize the hypothesis that cells have to undergo several transformations on their way to malignancy. This hypothesis assumes that a preneoplastic cell of a later stage arises through a mutational event of a single cell of a previous stage and that preneoplastic cells proliferate clonally. However, there is some evidence that multi-stage models cannot adequately describe the formation and the progression of preneoplastic lesions at least in certain organs [Math. Biosci. 168 (2000) 167]. An alternative model assuming that all cells in a colony of altered hepatocytes change their phenotype more or less simultaneously rather than by mutation of single cells has already been introduced [Math. Biosci. 148 (1998) 181] and is called color-shift model (CSM). This model assumed deterministic phenotype-independent growth for the foci once they are generated. An expansion of the CSM allowing for variability between deterministic growth behaviour of phenotypically different colonies is presented (modCSM) and the model is applied to focal lesion data from a rat hepatocarcinogenesis experiment. The fit of the originally proposed and the modCSM are compared with respect to their ability to predict numbers and radii of preneoplastic cell foci.     

Hormonal and hormone-like effects eliciting hepatocarcinogenesis

In various species, the manifestation of hepatocellular neoplasms is regularly preceded by preneoplastic foci of altered hepatocytes (FAH), the cellular phenotype of which is strikingly similar in experimental and human hepatocarcinogenesis, irrespective of the etiology of this process. The different types of FAH have been related to three main preneoplastic hepatocellular lineages: 1) the glycogenotic-basophilic cell lineage, 2) its xenomorphic-tigroid cell variant, and 3) the amphophilic-basophilic cell lineage. The predominant glycogenotic-basophilic and tigroid cell lineages developed especially after exposure to DNA-reactive chemicals, radiation, viruses, transgenic oncogenes and local hyperinsulinism. The early phenotypes of these lineages indicate an initiation by insulin or insulinomimetic effects of the oncogenic agents, triggering the raf-Map kinase signal transduction pathway. In contrast, the amphophilic-basophilic cell lineage has mainly been observed after exposure of rodents to not directly DNA-reactive peroxisome proliferators but also hepadnaviridae, its biochemical pattern mimiking an effect of thyroid hormone.     

Murine embryonic stem cell-derived hepatocytes correct metabolic liver disease after serial liver repopulation

Although embryonic stem (ES) cell-derived hepatocytes have the capacity for liver engraftment and repopulation, their in vivo hepatic function has not been analyzed yet. We aimed to determine the metabolic function and therapeutic action of ES cell-derived hepatocytes after serial liver repopulations in fumaryl acetoacetate hydrolase knockout (Fah(-/-)) mice. Albumin expressing (Alb(+)) cells were obtained by hepatic differentiation of ES cells using two frequently reported methods. After transplantation, variable levels of liver repopulation were found in Fah(-/-) mice recipients. FAH expressing (FAH(+)) hepatocytes were found either as single cells or as nodules with multiple hepatocytes. After serial transplantation, the proportion of the liver that was repopulated by the re-transplanted FAH(+) hepatocytes increased significantly. ES cell-derived FAH(+) hepatocytes were found in homogenous nodules and corrected the liver metabolic disorder of Fah(-/-) recipients and rescued them from death. ES cell-derived hepatocytes had normal karyotype, hepatocytic morphology and metabolic function both in vitro and in vivo. In conclusion, ES cell-derived hepatocytes were capable of liver repopulation and correction of metabolic defects after serial transplantation. Our results are an important piece of evidence to support future clinical applications of ES cell-derived hepatocytes in treating liver diseases.     

Investigating the formation and growth of alpha-particle radiation-induced foci of altered hepatocytes: a model-based approach

The effect of alpha-particle radiation on the formation and increase in volume of preneoplastic liver lesions was investigated in an animal experiment. Mice were divided into four groups; two groups received different doses of the alpha-particle-labeled antibody (213)Bi-anti CD19 ((213)Bi-CD19), Thorotrast was administered to one group, and one group was left untreated. Hematoxylin and eosin-stained liver sections were evaluated for preneoplastic foci of altered hepatocytes 6, 12 and 17 months after treatment. The density and size distribution of focal transections were described by a mechanistic model for the formation and growth of foci of altered hepatocytes. The negative control and the (213)Bi-CD19 groups were combined to investigate the dose-response relationship for model parameters describing the formation and growth of foci of altered hepatocytes. Although (213)Bi-CD19 was given by single injection, the effect on formation of foci of altered hepatocytes lasted for the entire experiment. Likelihood-ratio tests comparing nested models showed that (213)Bi-CD19 increases the rates of both the formation and growth of foci of altered hepatocytes. Comparing the effects of Thorotrast with those of (213)Bi-CD19 revealed that Thorotrast had an effect similar to that of a low dose of (213)Bi-CD19, but the effect on focus formation was slightly smaller whereas the effect on focus growth was slightly higher for Thorotrast, in contrast to a low dose of (213)Bi-CD19.     

Ploidy Reductions in Murine Fusion-Derived Hepatocytes

We previously showed that fusion between hepatocytes lacking a crucial liver enzyme, fumarylacetoacetate hydrolase (FAH), and wild-type blood cells resulted in hepatocyte reprogramming. FAH expression was restored in hybrid hepatocytes and, upon in vivo expansion, ameliorated the effects of FAH deficiency. Here, we show that fusion-derived polyploid hepatocytes can undergo ploidy reductions to generate daughter cells with one-half chromosomal content. Fusion hybrids are, by definition, at least tetraploid. We demonstrate reduction to diploid chromosome content by multiple methods. First, cytogenetic analysis of fusion-derived hepatocytes reveals a population of diploid cells. Secondly, we demonstrate marker segregation using ß-galactosidase and the Y-chromosome. Approximately 2–5% of fusion-derived FAH-positive nodules were negative for one or more markers, as expected during ploidy reduction. Next, using a reporter system in which ß-galactosidase is expressed exclusively in fusion-derived hepatocytes, we identify a subpopulation of diploid cells expressing ß-galactosidase and FAH. Finally, we track marker segregation specifically in fusion-derived hepatocytes with diploid DNA content. Hemizygous markers were lost by ≥50% of Fah-positive cells. Since fusion-derived hepatocytes are minimally tetraploid, the existence of diploid hepatocytes demonstrates that fusion-derived cells can undergo ploidy reduction. Moreover, the high degree of marker loss in diploid daughter cells suggests that chromosomes/markers are lost in a non-random fashion. Thus, we propose that ploidy reductions lead to the generation of genetically diverse daughter cells with about 50% reduction in nuclear content. The generation of such daughter cells increases liver diversity, which may increase the likelihood of oncogenesis.     

Obstructive cholestasis induces TNF-alpha- and IL-1 -mediated periportal downregulation of Bsep and zonal regulation of Ntcp, Oatp1a4, and Oatp1b2

Inverse acinar regulation of Mrp2 and 3 represents an adaptive response to hepatocellular cholestatic injury. We studied whether obstructive cholestasis (bile duct ligation) and LPS treatment affect the zonal expression of Bsep (Abcb11), Mrp4 (Abcc4), Ntcp (Slc10a1), and Oatp isoforms (Slco1a1, Slco1a4, and slco1b2) in rat liver, as analyzed by semiquantitative immunofluorescence. Contribution of TNF-alpha and IL-1beta to transporter zonation in obstructive cholestasis was studied by cytokine inactivation. In normal liver Bsep, Mrp4, Ntcp, and Oatp1a1 were homogeneously distributed in the acinus, whereas Oatp1a4 and Oatp1b2 expression increased from zone 1 to 3. Glutamine synthetase-positive pericentral hepatocytes exhibited markedly lower Oatp1a4 expression than the remaining zone 3 hepatocytes. In cholestatic liver Bsep and Ntcp immunofluorescence in periportal hepatocytes significantly decreased to 66 +/- 4% (P < 0.01) and 67 +/- 7% (P < 0.05), whereas it was not altered in pericentral hepatocytes. Oatp1a4 was significantly induced in hepatocytes with a primarily low expression, i.e., in periportal hepatocytes and in glutamine synthetase-positive pericentral hepatocytes. Likewise, Oatp1b2 was upregulated in periportal hepatocytes. Mrp4 zonal induction was homogeneous. Inactivation of TNF-alpha and IL-1beta prevented periportal downregulation of Bsep. Recruitment of neutrophils and polymorphonuclear cells mainly occurred in the periportal zone. Likewise, IL-1beta induction was largely found periportally. No significant transporter zonation was seen following LPS treatment. In conclusion, zonal downregulation of Bsep in obstructive cholestasis is associated with portal inflammation and is mediated by TNF-alpha and IL-1beta. Periportal downregulation of Ntcp and induction of Oatp1a4 and Oatp1b2 may represent adaptive mechanisms to reduce cholestatic injury in hepatocytes with profound downregulation of Bsep and Mrp2.     

Crystal structure and mechanism of a carbon-carbon bond hydrolase.

BACKGROUND: Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of tyrosine and phenylalanine catabolism, the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate, to yield fumarate and acetoacetate. FAH has no known sequence homologs and functions by an unknown mechanism. Carbon-carbon hydrolysis reactions are essential for the human metabolism of aromatic amino acids. FAH deficiency causes the fatal metabolic disease hereditary tyrosinemia type I. Carbon-carbon bond hydrolysis is also important in the microbial metabolism of aromatic compounds as part of the global carbon cycle. RESULTS: The FAH crystal structure has been determined by rapid, automated analysis of multiwavelength anomalous diffraction data. The FAH polypeptide folds into a 120-residue N-terminal domain and a 300-residue C-terminal domain. The C-terminal domain defines an unusual beta-strand topology and a novel 'mixed beta-sandwich roll' structure. The structure of FAH complexed with its physiological products was also determined. This structure reveals fumarate binding near the entrance to the active site and acetoacetate binding to an octahedrally coordinated calcium ion located in close proximity to a Glu-His dyad. CONCLUSIONS: FAH represents the first structure of a hydrolase that acts specifically on carbon-carbon bonds. FAH also defines a new class of metalloenzymes characterized by a unique alpha/beta fold. A mechanism involving a Glu-His-water catalytic triad is suggested based on structural observations, sequence conservation and mutational analysis. The histidine imidazole group is proposed to function as a general base. The Ca(2+) is proposed to function in binding substrate, activating the nucleophile and stabilizing a carbanion leaving group. An oxyanion hole formed from sidechains is proposed to stabilize a tetrahedral alkoxide transition state. The proton transferred to the carbanion leaving group is proposed to originate from a lysine sidechain. The results also reveal the molecular basis for mutations causing the hereditary tyrosinemia type 1.     

The Caenorhabditis elegans K10C2.4 gene encodes a member of the fumarylacetoacetate hydrolase family: a Caenorhabditis elegans model of type I tyrosinemia

In eukaryotes and many bacteria, tyrosine is degraded to produce energy via a five-step tyrosine degradation pathway. Mutations affecting the tyrosine degradation pathway are also of medical importance as mutations affecting enzymes in the pathway are responsible for type I, type II, and type III tyrosinemia. The most severe of these is type I tyrosinemia, which is caused by mutations affecting the last enzyme in the pathway, fumarylacetoacetate hydrolase (FAH). So far, tyrosine degradation in the nematode Caenorhabditis elegans has not been studied; however, genes predicted to encode enzymes in this pathway have been identified in several microarray, proteomic, and RNA interference (RNAi) screens as perhaps being involved in aging and the control of protein folding. We sought to identify and characterize the genes in the worm tyrosine degradation pathway as an initial step in understanding these findings. Here we describe the characterization of the K10C2.4, which encodes a homolog of FAH. RNAi directed against K10C2.4 produces a lethal phenotype consisting of death in young adulthood, extensive damage to the intestine, impaired fertility, and activation of oxidative stress and endoplasmic stress response pathways. This phenotype is due to alterations in tyrosine metabolism as increases in dietary tyrosine enhance it, and inhibition of upstream enzymes in tyrosine degradation with RNAi or genetic mutations reduces the phenotype. We also use our model to identify genes that suppress the damage produced by K10C2.4 RNAi in a pilot genetic screen. Our results establish worms as a model for the study of type I tyrosinemia.     

Beneficial impact of L-carnitine in liver: a study in a rat model of syndrome X

The present study was designed to explore whether L-carnitine (CA) regulates insulin signaling and modulates the changes in liver in a well-characterized insulin resistant rat model. Adult male Wistar rats were divided into 4 groups. Groups I and IV animals received starch-based control diet, while groups II and III rats were fed a high fructose-diet (60 g/100 g). Groups III and IV animals additionally received CA (300 mg/kg/day i.p). After a period of 60 days hepatic tyrosine phosphorylation status was determined by assaying protein tyrosine phosphatase (PTP) and protein tyrosine kinase (PTK) activities. Oxidative damage was monitored by immunohistochemical localization of 4-hydroxynonenal (4-HNE), 3-nitrotyrosine (3-NT) and dinitrophenol (DNP)-protein adducts. In addition protein kinase C beta II (PKC beta II) expression, propidium iodide staining of isolated hepatocytes and histology of liver tissue were determined to examine liver integrity. Fructose-fed rats displayed reduced insulin action, increased expression of PKC beta II, altered histology, fragmentation of hepatocyte nuclear DNA, and accumulation of oxidatively modified proteins. Simultaneous treatment with CA alleviated the abnormalities associated with fructose feeding. In summary the data suggest that elevated oxidative damage and PKC expression could in part induce insulin resistance and CA has beneficial impact on liver during insulin resistance with modulatory effects at the post-receptor level.     

New modified Square Root and Schoolfield models for predicting bacterial growth rate as a function of temperature

Summary A new modified Square Root model and two new modified Schoolfield models were evaluated for their ability to predict the growth rate ofYersinia enterocolitica as a function of temperature. The new Square Root model fits the data better than both the original Square Root model and the Zwietering Square Root model. Both new Schoolfield models, a six-and a four-parameter equation, fit the data better than the original Schoolfield model. The new four-parameter Schoolfield model was developed by removing the term describing low temperature inactivation from the new six-parameter Schoolfield model. Inclusion of the two extra parameters in the new six-parameter Schoolfield model (F=318) did not significantly improve the fit compared to the new fourparameter Schoolfield model (F=488).     

On a property of the exponential distribution

If t is an independent exponentially distributed random variable, the distribution p = [t - x] is a modified geometric distribution, similar to the result of HAWKINS and KOTZ (1976), x is uniform.     

Structural and functional analysis of missense mutations in fumarylacetoacetate hydrolase, the gene deficient in hereditary tyrosinemia type 1

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by a deficiency of the enzyme involved in the last step of tyrosine degradation, fumarylacetoacetate hydrolase (FAH). Thus far, 34 mutations in the FAH gene have been reported in various HT1 patients. Site-directed mutagenesis of the FAH cDNA was used to investigate the effects of eight missense mutations found in HTI patients on the structure and activity of FAH. Mutated FAH proteins were expressed in Escherichia coli and in mammalian CV-1 cells. Mutations N16I, F62C, A134D, C193R, D233V, and W234G lead to enzymatically inactive FAH proteins. Two mutations (R341W, associated with the pseudo-deficiency phenotype, and Q279R) produced proteins with a level of activity comparable to the wild-type enzyme. The N16I, F62C, C193R, and W234G variants were enriched in an insoluble cellular fraction, suggesting that these amino acid substitutions interfere with the proper folding of the enzyme. Based on the tertiary structure of FAH, on circular dichroism data, and on solubility measurements, we propose that the studied missense mutations cause three types of structural effects on the enzyme: 1) gross structural perturbations, 2) limited conformational changes in the active site, and 3) conformational modifications with no significant effect on enzymatic activity.     

A minimal model of non-hyperbolic enzyme and receptor kinetics

A simplified version of P.W. Kühl's Recovery Model [Biochem. J. 298 (1994) 171-180] has been developed in which the duration of the recovery phase of receptor or enzyme (macro)molecule was assumed to be a random value distributed exponentially like other model parameters. The model has been shown to retain all the properties of the original Recovery Model except for its ability to yield asymmetric dose-response curves (if plotted in semi-logarithmic scale). Due to its simplicity, the present model is applicable for routine fitting to experimental data. In enzyme kinetics, the model yields a diversity of non-hyperbolic dose-response curves both with higher and lower steepness than that of Henri-type ones. In receptor kinetics, the diversity of dose-response curves is further increased due to virtually no restraints being imposed on the efficacies of any state of the macromolecule.     

Final height in children with idiopathic growth hormone deficiency treated with a fixed dose of recombinant growth hormone

There is no consensus regarding the optimal dosing of recombinant human growth hormone (rhGH) for children with growth hormone deficiency (GHD). Our objective was to evaluate the final adult height (FAH) in children with idiopathic GHD treated with a fixed rhGH dose of 0.18 mg/kg/week. We reviewed all charts of patients with idiopathic GHD treated with rhGH since 1985 who reached FAH. Ninety-six patients were treated for an average of 5.4 years. The mean age was 11.9 years, the mean height -2.87 standard deviation score (SDS) and the mean FAH was -1.04 SDS. Females had a lower predicted adult height than males at the initiation of therapy (-2.0 vs. -1.01 SDS; p = 0.0087) but a higher FAH - predicted adult height (1.08 vs. 0.04 SDS; p = 0.0026). In multiple regression analysis, the FAH SDS was positively related to the midparental height SDS, the height SDS at GH initiation and growth velocity during the first year of therapy, and negatively correlated with peak GH and bone age at initiation (r(2) = 0.51; p < 0.005). Treatment of children with idiopathic GHD with a fixed dose of 0.18 mg/kg/week rhGH is sufficient to reach FAH within 2 SDS of the normal population range (84%) with an average FAH within -0.5 SDS of midparental height.     

Offering a forage crop at pasture did not adversely affect voluntary cow traffic or milking visits in a pasture-based automatic milking system

Feed is a strong incentive for encouraging cows in automatic milking systems (AMS) to voluntarily move around the farm and achieve milkings distributed across the 24 h day. It has been reported that cows show preferences for some forages over others, and it is possible that offering preferred forages may increase cow traffic. A preliminary investigation was conducted to determine the effect of offering a forage crop for grazing on premilking voluntary waiting times in a pasture-based robotic rotary system. Cows were offered one of two treatments (SOYBEAN or GRASS) in a cross-over design. A restricted maximum likelihood procedure was used to model voluntary waiting times. Mean voluntary waiting time was 45.5±6.0 min, with no difference detected between treatments. High and mid-production cows spent <44 min/milking in the premilking yard compared with >55 min/milking for low-production cows, whereas waiting time increased as queue length increased. Voluntary waiting time was 23% and 80% longer when cows were fetched from the paddock or had a period of forced waiting before volunteering for milking, respectively. The time it took cows to return to the dairy since last exiting was not affected by treatment, with a mean return time of 13.7±0.6 h. Although offering SOYBEAN did not encourage cows to traffic more readily through the premilking yard, the concept of incorporating forage crops in AMS still remains encouraging if the aim is to increase the volume or quantity of home-grown feed rather than improving cow traffic.     

Maleylacetoacetate isomerase (MAAI/GSTZ)-deficient mice reveal a glutathione-dependent nonenzymatic bypass in tyrosine catabolism

In mammals, the catabolic pathway of phenylalanine and tyrosine is found in liver (hepatocytes) and kidney (proximal tubular cells). There are well-described human diseases associated with deficiencies of all enzymes in this pathway except for maleylacetoacetate isomerase (MAAI), which converts maleylacetoacetate (MAA) to fumarylacetoacetate (FAA). MAAI is also known as glutathione transferase zeta (GSTZ1). Here, we describe the phenotype of mice with a targeted deletion of the MAAI (GSTZ1) gene. MAAI-deficient mice accumulated FAA and succinylacetone in urine but appeared otherwise healthy. This observation suggested that either accumulating MAA is not toxic or an alternate pathway for MAA metabolism exists. A complete redundancy of MAAI could be ruled out because substrate overload of the tyrosine catabolic pathway (administration of homogentisic acid, phenylalanine, or tyrosine) resulted in renal and hepatic damage. However, evidence for a partial bypass of MAAI activity was also found. Mice doubly mutant for MAAI and fumarylacetoacetate hydrolase (FAH) died rapidly on a normal diet, indicating that MAA could be isomerized to FAA in the absence of MAAI. Double mutants showed predominant renal injury, indicating that this organ is the primary target for the accumulated compound(s) resulting from MAAI deficiency. A glutathione-mediated isomerization of MAA to FAA independent of MAAI enzyme was demonstrated in vitro. This nonenzymatic bypass is likely responsible for the lack of a phenotype in nonstressed MAAI mutant mice.