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Acrylamide (AA), classified as class 2A carcinogen (probably carcinogenic to humans) by the International Agency for Research on Cancer (IARC), is formed during heating of food from reducing carbohydrates and asparagine by Maillard reaction chemistry. After dietary uptake, AA is in part metabolically converted into the proximate genotoxic phase I metabolite glycidamide (GA). GA reacts with nucleophilic base positions in DNA, primarily forming N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) adducts. In a competing phase II biotransformation pathway AA, as well as its phase I metabolite GA, is coupled to glutathione (GSH). The GSH coupling products are further biotransformed and excreted via urine as mercapturic acids (MA), N-acetyl-S-(2-carbamoylethyl)cysteine (AAMA), and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine (GAMA). In the present study, hepatic biotransformation pathways and DNA adduct formation were studied in primary rat hepatocytes, incubated with AA (0.2-2,000 μM) for up to 24 h. Contents of AA-GSH, GA, AAMA, and GAMA were measured in the cell culture medium after solid phase extraction (SPE). N7-GA-Gua adducts in DNA of hepatocytes were determined by HPLC-ESI-MS/MS after lysis of the cells and neutral thermal hydrolysis. Formation of AA-GSH was linear with AA concentration and incubation time and became detectable already at 0.2 μM (4 h). In contrast to AA, GA was not detected before 16 h incubation at 10-fold higher AA concentration (2 μM). In summary, the rate of AA-GSH formation was found to be about 1.5-3 times higher than that of GA formation. N7-GA-Gua adducts were found only at the highest AA concentration tested (2,000 μM).

Cultivated hepatocytes represent a well-established in vitro system. However, the applicability of hepatocytes in toxicogenomics is still controversially discussed. Recently, an in vivo/in vitro discrepancy has been described, whereby the non-genotoxic rat liver carcinogen methapyrilene alters the expression of the metabolizing genes SULT1A1 and ABAT, as well as the DNA damage response gene GADD34 in vitro, but not in vivo. If the collagen sandwich cultures of hepatocytes really produce false-positive data, this would compromise its application in toxicogenomics. To revisit the putative in vivo/in vitro discrepancy, we first analyzed and modeled methapyrilene concentrations in the portal vein of rats. The relatively short half-life of 2.8 h implies a rapid decrease in orally administered methapyrilene in vivo below concentrations that can cause gene expression alterations. This corresponded to the time-dependent alteration levels of GADD34, ABAT and SULT1A1 RNA in the liver: RNA levels are altered 1, 6 and 12 h after methapyrilene administration, but return to control levels after 24 and 72 h. In contrast, methapyrilene concentrations in the culture medium supernatant of primary rat hepatocyte cultures decreased slowly. This explains why GADD34, ABAT and SULT1A1 were still deregulated after 24 h exposure in vitro, but not in vivo. It should also be considered that the earliest analyzed time point in the previous in vivo studies was 24 h after methapyrilene administration. In conclusion, previously observed in vitro/in vivo discrepancy can be explained by different pharmacokinetics present in vitro and in vivo. When the in vivo half-life is short, levels of some initially altered genes may have returned to control levels already 24 h after administration.

The piperazine derivatives most frequently consumed for recreational purposes are 1-benzylpiperazine, 1-(3,4-methylenedioxybenzyl) piperazine, 1-(3-trifluoromethylphenyl) piperazine and 1-(4-methoxyphenyl) piperazine. Generally, they are consumed as capsules, tablets or pills but also in powder or liquid forms. Currently, the precise mechanism by which piperazine designer drugs induce hepatotoxicity and whether they act by a common pathway is unclear. To answer this question, we performed a gene array study with rat hepatocytes incubated with the four designer drugs. Non-cytotoxic concentrations were chosen that neither induce a decrease in reduced glutathione or ATP depletion. Analysis of the gene array data showed a large overlap of gene expression alterations induced by the four drugs. This ‘piperazine designer drug consensus signature’ included 101 up-regulated and 309 down-regulated probe sets (p

Although cultivated hepatocytes are widely used in the studies of drug metabolism, their application in toxicogenomics is considered as problematic, because previous studies have reported only little overlap between chemically induced gene expression alterations in liver in vivo and in cultivated hepatocytes. Here, we identified 22 genes that were altered in livers of rats after oral administration of the liver carcinogens aflatoxin B1 (AB1), 2-nitrofluorene (2-NF), methapyrilene (MP) or piperonyl-butoxide (PBO). The functions of the 22 genes have been classified into two groups. Genes related to stress response, DNA repair or metabolism and genes associated with cell proliferation, respectively. Next, rat hepatocyte sandwich cultures were exposed to AB1, 2-NF, MP or PBO for 24h and expression of the above mentioned genes was determined by RT-qPCR. Significant correlations between the degree of gene expression alterations in vivo and in vitro were obtained for the stress, DNA repair and metabolism associated genes at concentrations covering a range from cytotoxic concentrations to non-toxic/in vivo relevant concentrations. In contrast to the stress associated genes, no significant in vivo/in vitro correlation was obtained for the genes associated with cell proliferation. To understand the reason of this discrepancy, we compared replacement proliferation in vivo and in vitro. While hepatocytes in vivo, killed after administration of hepatotoxic compounds, are rapidly replaced by proliferating surviving cells, in vitro no replacement proliferation as evidenced by BrdU incorporation was observed after washing out hepatotoxic concentrations of MP. In conclusion, there is a good correlation between gene expression alterations induced by liver carcinogens in vivo and in cultivated hepatocytes. However, it should be considered that cultivated primary hepatocytes do not show replacement proliferation explaining the in vivo/in vitro discrepancy concerning proliferation associated genes.

Our study was performed in the context of an in vitro primary hepatic cell culture as an alternative for the in vivo cancerogenic bioassay. The 29 substances which are to be used in the in vitro primary hepatic cell culture have been tested in 2-year bioassays and a 14-day short term study. The aim of this modelling study was to simulate the concentration--time profile of the compounds when given by the oral route at the doses tested in the previous studies taking into account the percentage of the dose absorbed. The model contained seven tissue compartments with uptake from the gastrointestinal tract into the portal vein. Because the primary hepatic cell culture is metabolically competent and the primary interest was to model the concentration in the portal vein, the hepatic vein and the systemic circulation (blood) in the beginning we did not include elimination. Partitioning between blood and tissues was calculated according to a published biologically based algorithm. The substances' kinetic profile differed according to their blood: tissue partitioning. Maximal concentrations in portal vein, hepatic vein and the blood depended mainly on the dose and the fraction absorbed which were the most critical parameters in this respect. Our study demonstrates an application of BPTK modelling for the purpose to simulate concentrations for planning the doses for an in vitro study. BPTK modelling seems to be a better approach than using data from in vitro studies on cytotoxicity.

Hepatocytes in culture are a valuable tool to investigate mechanisms involved in the response of the liver to cytokines. However, it is well established that hepatocytes cultured as monolayers on dried stiff collagen dedifferentiate, loosing specialized liver functions. In contrast, softer matrix systems like gelled collagen help to preserve these structural and functional features. We show that the de-differentiation process induced in conventional dry collagen is a reversible consequence of a specific signaling network constellation triggered by the extracellular matrix that results in apoptosis resistance. A dried stiff collagen activates Akt and ERK1/2 pathways that results in apoptosis resistance. In contrast to stiff collagen, a soft collagen gel does not activate these pathways keeping the hepatocytes in a state where they remain sensitive to TGF-beta-induced apoptosis. Finally, we show that matrix-induced apoptosis resistance is reversible by re-plating cells from dried stiff to soft gel collagen. Practical consequences of these observations are that differentiated functions of hepatocytes, such as metabolism, endocytosis, and apoptosis, should be studied in hepatocyte sandwiches. On the other hand, proliferation and regeneration associated signaling can better be studied in hepatocytes cultured on collagen monolayers. In this chapter we focus on mechanisms that influence apoptosis sensitivity in cultured mouse hepatocytes.

Hepatocytes in culture are a valuable tool to investigate mechanisms involved in the response of the liver to cytokines. However, it is well established that hepatocytes cultured as monolayers on dried stiff collagen dedifferentiate, loosing specialized liver functions. In contrast, softer matrix systems like gelled collagen help to preserve these structural and functional features. We show that the de-differentiation process induced in conventional dry collagen is a reversible consequence of a specific signaling network constellation triggered by the extracellular matrix that results in apoptosis resistance. A dried stiff collagen activates Akt and ERK1/2 pathways that results in apoptosis resistance. In contrast to stiff collagen, a soft collagen gel does not activate these pathways keeping the hepatocytes in a state where they remain sensitive to TGF-beta-induced apoptosis. Finally, we show that matrix-induced apoptosis resistance is reversible by re-plating cells from dried stiff to soft gel collagen. Practical consequences of these observations are that differentiated functions of hepatocytes, such as metabolism, endocytosis, and apoptosis, should be studied in hepatocyte sandwiches. On the other hand, proliferation and regeneration associated signaling can better be studied in hepatocytes cultured on collagen monolayers. In this chapter we focus on mechanisms that influence apoptosis sensitivity in cultured mouse hepatocytes.

Recently, epithelial to mesenchymal transition (EMT) has been shown to represent a feature of dedifferentiating hepatocytes in vitro. Three-dimensional soft collagen gels can antagonize but not completely abolish this effect. Hormonal additives to culture media are known to maintain differentiated hepatocyte functions. Therefore, we studied whether insulin and dexamethasone antagonize EMT in cultured hepatocytes. Both hormones antagonized but not completely abolished certain morphological features of EMT. Dexamethasone antagonized acquisition of fibroblastoid shape, whereas insulin favored bile canaliculi formation. In a subsequent step, we analyzed expression of a battery of EMT-related genes. Of all markers tested, vimentin and snail-1 correlated best with morphological features of EMT. Interestingly, dexamethasone reduced expression levels of both vimentin and snail-1, whereas the influence of insulin was less pronounced. An important result of this study is that 12 out of 17 analyzed EMT markers were transcriptionally influenced by dexamethasone (vimentin, snail-1, snail-2, HNF4α, Twist-1, ZEB2, fibronectin, occludin, MMP14, claudin-1, cytokeratin-8, and cytokeratin-18), whereas the remaining factors seemed to be less dependent on dexamethasone. In conclusion, EMT markers in hepatocytes can be classified as dexamethasone-dependent versus -independent.

Recent studies have presented evidence that in vivo obtained gene expression data can be used for carcinogen classification, for instance to differentiate between genotoxic and non-genotoxic carcinogens. However, although primary rat hepatocytes represent a well-established in vitro system for drug metabolism and enzyme induction, they have not yet been systematically optimized for toxicogenomic studies. The latter may be confounded by the fact that cultured hepatocytes show strong spontaneous alterations in gene expression patterns. Therefore, we addressed the following questions: (1) which culture system is optimal, comparing sandwich, Matrigel and 2D cultures, (2) how critical is the impact of culture period on substance-induced alterations in gene expression and (3) do these substance-induced alterations in cultured hepatocytes occur already at in vivo relevant concentrations? For this purpose we analyzed the expression of four genes, namely Abat, Gsk3beta, Myd116 and Sult1a1 that recently have been reported to be influenced by the antihistamine and non-genotoxic carcinogen methapyrilene (MPy). The most reproducible effects of MPy were observed in sandwich cultures. Induction factors of Gsk3beta and Myd116 at 100 microM MPy were 2 and 4 (medians), respectively, whereas expression of Abat and Sult1a1 were inhibited by factors of 7 and 5, respectively. Similar results were observed in hepatocytes maintained for 24 h or 3 weeks in sandwich culture with respect to the influence of MPy on the expression of Abat, Gsk3beta, Myd116 and Sult1a1. To determine whether MPy influences gene expression at in vivo relevant concentrations, 3.5 mg/kg MPy were administered to male Wistar rats intraperitoneally, resulting in plasma concentrations ranging between 1.72 and 0.32 microM 5 and 80 min after injection. Inhibition of Abat and Sult1a1 expression in vitro already occurred at in vivo relevant concentrations of 0.39 microM MPy. Induction of Myd116 was observed at 6.25 microM which is higher but in the same order of magnitude as in vivo relevant concentrations. In conclusion, the presented data strongly suggest that sandwich cultures are most adequate for detection of MPy-induced gene expression alterations and the effect of MPy was detected at in vivo relevant concentrations.

Approximately 5,000 of 6 million annual visitors of the Oktoberfest in Munich have to undergo medical treatment. Patients with alcohol intoxication without trauma or further complications are all treated in a specialized medical camp. We studied these patients in order to identify risk factors and to assess the relevance of the Glasgow Coma Score (GCS) and of ethanol blood concentrations for patient management. In 2004 totally 405 patients suffering from ethanol intoxication without trauma were treated in the medical camp. A complete set of the following data was obtained from all 405 patients: GCS, ethanol blood concentration, age, sex, blood pressure (mean, systolic and diastolic), body temperature, heart rate, blood sugar, GOT, γ-GT, and CK. A multivariate logistic regression model was applied to identify risk factors predicting patients at increased risk of hospitalization. Low GCS (≤8 vs. >8, OR: 4.18, CI: 1.96–8.65) low age (20–29 vs. ≥30 years, OR: 2.35, CI: 1.05–5.65) and male gender (male vs. female, OR: 3.58, CI: 1.36–9.34) independently predicted patients that had to be hospitalized. All other parameters including ethanol blood concentrations were not explanatory. Patients with GCS ≤ 8 (n = 66) had a lower median blood pressure (P = 0.0312) and showed a smaller increase in blood pressure during the observation period compared to patients with GCS > 8 (P

Recent studies have presented evidence that in vivo obtained gene expression data canbe used for carcinogen classification, for instance to differentiate between genotoxic andnon-genotoxic liver carcinogens. This led several groups to study RNA-expressionpatterns also in cultured hepatocytes. However, these experiments resulted in conflictingdata to the in vivo situation. An example are the genes Gsk3â and Myd116 that have beenreported to be deregulated by methapyrilene (MPy) in hepatocytes in vitro but not in ratliver in vivo . To address this discrepancy, we first optimized an in vitro system with culturedhepatocytes. Testing various culture conditions (collagen coated dishes vs. Matrigel vs.sandwich cultures) and medium additives, we finally decided to use an in vitro system withchemically defined serum free culture medium and hepatocytes between two collagenlayers (sandwich culture). Interestingly, the reason for the in vivo / in vitro discrepancy couldclearly be identified. MPy in rats has a very short half-life of approx. 2.8 h. Therefore, weobserved an induction of Gsk3â and Myd116 after 13 and 18 h that decreased to basallevels after 24 h. Obviously, MPy caused only a transient induction. However, in vitro apermanent induction was obtained due to the constant concentration of MPy in the culturemedium. In conclusion the reported difference between in vivo and in vitro data was aconsequence of pharmacokinetics and not of qualitatively different behaviour ofhepatocytes in vitro and in vivo .