There is convincing evidence that cellular prooxidant states--that is, increased concentrations of active oxygen and organic peroxides and radicals--can promote initiated cells to neoplastic growth. Prooxidant states can be caused by different classes of agents, including hyperbaric oxygen, radiation, xenobiotic metabolites and Fenton-type reagents, modulators of the cytochrome P-450 electron-transport chain, peroxisome proliferators, inhibitors of the antioxidant defense, and membrane-active agents. Many of these agents are promoters or complete carcinogens. They cause chromosomal damage by indirect action, but the role of this damage in carcinogenesis remains unclear. Prooxidant states can be prevented or suppressed by the enzymes of the cellular antioxidant defense and low molecular weight scavenger molecules, and many antioxidants are antipromoters and anticarcinogens. Finally, prooxidant states may modulate the expression of a family of prooxidant genes, which are related to cell growth and differentiation, by inducing alterations in DNA structure or by epigenetic mechanisms, for example, by polyadenosine diphosphate-ribosylation of chromosomal proteins.

https://science.sciencemag.org/content/sci/227/4685/375.full.pdf

Prooxidant states and tumor promotion.

Commercial polychlorinated biphenyls (PCBs) and environmental extracts contain complex mixtures of congeners that can be unequivocally identified and quantitated. Some PCB mixtures elicit a spectrum of biochemical and toxic responses in humans and laboratory animals and many of these effects resemble those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons, which act through the aryl hydrocarbon (Ah)-receptor signal transduction pathway. Structure-activity relationships developed for PCB congeners and metabolites have demonstrated that several structural classes of compounds exhibit diverse biochemical and toxic responses. Structure-toxicity studies suggest that the coplanar PCBs, namely, 3,3',4,4'-tetrachlorobiphenyl (tetraCB), 3,3',4,4',5-pentaCB, 3,3',4,4',5,5'-hexaCB, and their monoortho analogs are Ah-receptor agonists and contribute significantly to the toxicity of the PCB mixtures. Previous studies with TCDD and structurally related compounds ...

Polychlorinated Biphenyls (PCBs): Environmental Impact, Biochemical and Toxic Responses, and Implications for Risk Assessment

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

/pdf/recent-advances-in-2d-and-3d-in-vitro-systems-using-primary-46n7hrqknx.pdf

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

The aryl hydrocarbon (Ah) receptor has occupied the attention of toxicologists for over two decades. Interest arose from the early observation that this soluble protein played key roles in the adaptive metabolic response to polycyclic aromatic hydrocarbons and in the toxic mechanism of halogenated dioxins and dibenzofurans. More recent investigations have provided a fairly clear picture of the primary adaptive signaling pathway, from agonist binding to the transcriptional activation of genes involved in the metabolism of xenobiotics. Structure-activity studies have provided an understanding of the pharmacology of this receptor; recombinant DNA approaches have identified the enhancer sequences through which this factor regulates gene expression; and functional analysis of cloned cDNAs has allowed the characterization of the major signaling components in this pathway. Our objective is to review the Ah receptor's role in regulation of xenobiotic metabolism and use this model as a framework for understanding the less well-characterized mechanism of dioxin toxicity. In addition, it is hoped that this information can serve as a model for future efforts to understand an emerging superfamily of related signaling pathways that control biological responses to an array of environmental stimuli.

Ah receptor signaling pathways

The Ah receptor (AHR) is a ligand-activated transcription factor that mediates a pleiotropic response to environmental contaminants such as benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. In an effort to gain insight into the physiological role of the AHR and to develop models useful in risk assessment, gene targeting was used to inactivate the murine Ahr gene by homologous recombination. Ahr-/- mice are viable and fertile but show a spectrum of hepatic defects that indicate a role for the AHR in normal liver growth and development. The Ahr-/- phenotype is most severe between 0-3 weeks of age and involves slowed early growth and hepatic defects, including reduced liver weight, transient microvesicular fatty metamorphosis, prolonged extramedullary hematopoiesis, and portal hypercellularity with thickening and fibrosis.

https://www.pnas.org/content/pnas/93/13/6731.full.pdf

Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development

In order to test the potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a promoter of hepatocarcinogenesis, rats which had received a single 10-mg/kg dose of diethylnitrosamine (DEN) following partial hepatectomy were given TCDD (0.14 and 1.4 micrograms/kg s.c. once every 2 weeks) for 7 months. Animals which received (a) only a single initiating dose of DEN after partial hepatectomy and no further treatment of (b) TCDD alone with no initiating dose of DEN exhibited relatively few enzyme-altered foci and no hepatocellular carcinomas. However, animals initiated with DEN and then given TCDD had a marked increase in enzyme-altered foci. At the higher dose of TCDD, hepatocellular carcinomas were present in five of seven rats. By means of three different enzyme markers used to evaluate the phenotypes of the enzyme-altered foci, a distinct phenotype heterogeneity of the foci was noted with a shift towards phenotypes exhibiting a greater deviation from normal liver when TCDD was given following DEN-partial hepatectomy. Quantitation of the numbers of enzyme-altered foci was performed by relating measurements made from two-dimensional tissue sections to the numbers of foci per unit volume of liver using relationships established in the field of stereology. The total volume of the liver occupied by the enzyme-altered foci, but not their number, increased with the dose of TCDD administered following DEN-partial hepatectomy. These studies demonstrate that TCDD is a potent promoting agent for hepatocarcinogenesis.

Quantitative Evaluation of the Promotion by 2,3,7,8-Tetrachlorodibenzo-p-dioxin of Hepatocarcinogenesis from Diethylnitrosamine

The mathematical science of quantitative stereology has established relationships for the quantitation of elements in three-dimensional space from observations on two-dimensional planes. This report describes the utilization and importance of such mathematical relationships for the quantitative analysis of focal hepatic lesions in terms relative to the volume of the liver.

Three examples are utilized to demonstrate the utility of such calculations in the three-dimensional quantitation of hepatic focal lesions. The first is that of a computer-simulated experiment based on defined hypothetical situations. The simulations demonstrate the applicability of the computations described in this report to the evaluation of two-dimensional data from typical animal experiments. The other two examples are taken from actual experiments and involve the transplantation of hepatic cell populations into the liver of suitably prepared hosts and the quantitation of altered foci produced by initiation with diethylnitrosamine-partial hepatectomy followed by promotion with phenobarbital. The quantitation of altered foci by means of a two-dimensional analysis (simple enumeration of focal intersections/area of tissue section) is proportional to the quantitation of foci per volume of liver provided that the mean diameter of the foci for each treatment is sufficiently uniform, as exemplified in the text by the transplantation experiment. When such mean diameters are unequal as in the diethylnitrosamine-phenobarbital experiment described herein, quantitation from three-dimensional analysis gives significantly different results as compared with enumeration of focal intersections on two-dimensional areas. These studies clearly demonstrate that the frequency and size of foci intersections viewed on two-dimensional tissue sections do not necessarily reflect the number or size of foci in the three-dimensional tissue. Only by quantitating the number and size of the foci in relation to the three-dimensional volume of the tissue can one determine the validity of the proportionality of data from two-dimensional measurements to the total number of foci per volume of tissue. Such a conclusion has important implications for quantitative studies on hepatocarcinogenesis as well as for the enumeration of premalignant lesions which occur during the natural history of carcinogenesis in any solid tissue.

https://cancerres.aacrjournals.org/content/canres/42/2/465.full.pdf

Application of Quantitative Stereology to the Evaluation of Enzymealtered Foci in Rat Liver

Studies on the hemorrhagic sweet clover disease iv. the isolation and crystallization of the hemorrhagic agent

The relative response to various initiating doses of diethylnitrosamine (DEN) and dimethylbenz[a]anthracene of the induction of numbers and size (vol. % of liver) of altered hepatic foci (AHF) in livers of adult female rats of the Sprague-Dawley and Fischer 344 (F-344) strains was studied by methods of quantitative stereology in the presence and absence of the promoting agent, phenobarbital (PB, 0.05% in the diet). In all cases, a relatively linear response with dose, even at the lowest doses employed, was obtained except for the numbers of AHF at the highest dose of DEN (30 mg/kg), which was not significantly different from that at a dose of 10 mg/kg in F-344 female rats. Similar dose-response data were obtained at various doses of two promoting agents effective in hepatocarcinogenesis, PB and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in livers of F-344 female rats following initiation with DEN (10 mg/kg) 24 h post-70% hepatectomy. The response to these agents exhibited threshold levels below which no increase in number or vol. % of liver of AHF was noted in comparison with that in livers of animals not treated with the promoting agents. At several subthreshold doses of both PB and TCDD an inhibition of AHF formation and growth (measured as vol. % of liver) was observed. Based on quantitative stereologic calculations, parameters for the estimation for the relative potency of chemicals as initiating or promoting agents have been established. These are defined as: initiation index = no. of foci induced X liver-1 X [mmol/kg body wt]-1 and promotion index = Vf/Vc X mmol-1 X weeks-1, where Vf is the total volume fraction (%) occupied by AHF in the livers of rats treated with the test agent and Vc is the total volume of AHF in control animals which have only been initiated. These parameters were calculated for a number of agents based on data published in the literature and from those reported herein. Neither parameter varied significantly with the dose of the initiating agent based on the data in this paper. The range of promotion indices extended over more than eight orders of magnitude, whereas that of initiation indices was much less variable. Such parameters may be useful as quantitative estimates of the potency of hepatocarcinogenic agents, such values having potential application to risk estimations.

A method to quantitate the relative initiating and promoting potencies of hepatocarcinogenic agents in their dose-response relationships to altered hepatic foci.

1. The hemorrahgic agent is spoiled sweet clover hay (Melilotus alaba) has been isolatd in a pure state, m. p. 288-289°, from experimentally produced spoiled hays as well as form hyas that killed cattle in argicultural practice.

2. This substance has the empirical formula C12H12O6. It is optically inactive. There are two acidic hydroxyls in the molecule. The acidity of the pure substance falls between that of the phenols and the carboxylic acids. A crystalline dimethyl other C12H12O4(OCH2)2 with a melting point fo 168-170° (physiologically inactive) has been prepared by methylation with diazomethane.

3. In the pure state the substance has a low soulubility in the ordinary organic solvents. It is insoluble in acid media. Basic solvenets. and dilute alkali effect solution radily (salt formation).

4. The substance could not be characterized or identified on the basis of its behavior towards the usual identification reagents. Its occurrence in nature has not previosly been reproted.

5. 1.5 mg. of hte crystalline hemorrhagic agent cause approximately the same reduciton in the prothrombin level or activity of the plasma of standardized susceptible reabbits in 40 hours as 50 gm. of the standard spoiled hay sample.

6. Spoiled sweet cover hays produced experimentally from Melilotus alba and those relized in agricultural parctice contain approximately 0.003 per cent of the hemorrhagic agent on the dry substance basis. The over-all yield of the subsance in a fractionation scheme involving sixteen steps approximates 86 per cent of the quantity present.

Harold A. Campbell

Papers

Quantitative Evaluation of the Promotion by 2,3,7,8-Tetrachlorodibenzo-p-dioxin of Hepatocarcinogenesis from Diethylnitrosamine

Application of Quantitative Stereology to the Evaluation of Enzymealtered Foci in Rat Liver

Studies on the hemorrhagic sweet clover disease iv. the isolation and crystallization of the hemorrhagic agent

A method to quantitate the relative initiating and promoting potencies of hepatocarcinogenic agents in their dose-response relationships to altered hepatic foci.

Studies on the hemorrhagic sweet clover disease iv. the isolation and crystallization of the hemorrhagic agent