Genetic and Environmental Toxicology

The metabolism of xenobiotic substances occurs primarily in the liver via oxidations (cytochromes P-450) and conjugation reactions (i.e. with glutathione), and is called biotransformation. The metabolites generated should be more water soluble than the parent compounds and thus more readily excretable. In many cases, however, reactive metabolites are formed especially via the oxidative metabolism. These, for example epoxides, are capable of binding to DNA and as a consequence can lead to mutations. Examples of mutagens/carcinogens acting this way are aflatoxins, nitrosamines, polycyclic aromatic hydrocarbons such as benzo(a)pyrene, etc.

 

Mutagenicity testing

Since the site of formation of reactive metabolites should also be the site of primary damage, we developed a mutagenicity test with primary rat hepatocytes based on the endpoints sister chromatid exchanges= SCE, chromosomal aberrations and micronuclei. Compared to tests with mammalian and human cell lines even in the presence of liver microsomal fractions this test proved to be more sensitive - based on the endpoint SCE by a factor of at least 100. Due to this high sensitivity and the finding that DNA damage persists in the liver the following applications were developed: a) Testing of complex environmental mixtures such as surface and groundwater as well as aerosol samples, i.e. water samples from the Aral Sea b) Biomonitoring with feral rodents a) Dietary intake of mutagens b) Testing of antimutagenic effects of pure compounds or complex mixtures, i. e. herbal extracts

Oxidative Stress

The background rates of the analysed mutagenic effects in primary hepatocytes are significantly higher than those in established cell lines; based on the endpoint SCE this means a factor of 2 to 3. This difference could be demonstrated by us to be in part dependent on the diet. Mainly, however, oxidative stress in primary culture is responsible for this effect and is due to a change of the oxygen partial pressure from approximately 8% in vivo to approximately 17% in vitro. Supplementation of antioxidants such as the vitamines E and C therefore can reduce the background levels by almost 50%.

This high sensitivity towards oxidative stress appears not to depend on the excess of molecular oxygen itself but rather on lipid peroxidation products generated, especially 4-hydroxynonenal = HNE, since hepatocytes react more sensitive to it than other cells. The dose response relationships obtained with HNE are not linear. Instead, they represent saturation-type curves indicating that either there is a saturation of the damage induced or that adaptation to the stressor occurs.

Oxidative stress appears to play an important role in a variety of pathological events ranging from cancer to Alzheimer´s desease to tissue damages after stroke and ischemia. In the latter cases endothelial cells are thought to be important mediators. In cooperation with Hans Bauer from the Institute of Molecular Biology of the Austrian Academy of Sciences we were able to confirm the sensitivity of brain endothelial cells and astrocytes to HNE and hypoxia/reoxygenation. These investigations are currently extended to primary human astrocytes and astrocytoma cells in a cooperation with the Christian Doppler Clinics, Salzburg (Peter Strasser).

Antioxidants and the β-Carotene paradox

All aerobic forms of life maintain elaborate anti-free-radical defense systems, also known as antioxidant systems. Antioxidants are molecules which can interact with oxidising agents before other molecules are damaged. Every cell in the body has antioxidant enzymes, the specialty of which is reducing oxygen radicals and ROS.

As a second line of defense, the body makes use of vitamins and other nutrients to antagonize the oxygen radical effects. Among these ‘’nonenzymatic antioxidants’’, the major antioxidants are the lipid-soluble vitamins (vitamin E and carotenoids) and the water-soluble vitamin C.

Results obtained from experiments on animals indicate a cancer preventive effect of antioxidants. Antioxidants (β-carotene, vitamin C, E, and other substances), interact with free radicals and prevent the damages they may cause.

β-Carotene, a natural fat-soluble pigment, is a precursor of vitamin A (retinol) and is present in liver, egg yolk, milk, butter, spinach, carrots, squash, broccoli, yams, tomato, cantaloupe, peaches, grains, etc.. It is essential for normal growth and development.

Beside the beneficial effects of β-carotene, the epidemiological evidence for such a beneficial role of β-carotene could not be supported by several large scale, randomized supplementation trials. Strikingly, in two major trials cancer incidence was increased with β-carotene supplementation in both smokers and asbestos workers.

It was therefore hypothesized that heavy oxidative stress leads to degradation of β-carotene giving rise to the formation of high amounts of cleavage products with prooxidant properties. These cleavage products were tested in the primary rat hepatocyte assay and proved to have a genotoxic potential even at concentrations which can be achieved in vivo. Furthermore, β-carotene supplementation during oxidative stress, i.e. hypoxia/reoxygenation leads to a dose-dependent increase of genotoxic effects compared to standard culture conditions. Currently, a project funded by the FWF addresses the genotoxic potential of β-carotene in primary rat and human pneumocytes, and the nature of the cleavage products formed apart from those already identified.


Isolation and characterisation of apoptosis inclucing factors

In the normal liver, apoptosis is a rare event. However, it has been shown that apoptosis can be induced by a variety of environmental conditions (e.g. acute ischemia, reoxygenation after hypoxia, intoxications) but also by the activity of growth inhibitory factors such as TGF 1 , TNF or activation of the Fas pathway.

We have made the observation, that a conditioned medium which had been collected after the first three hours of primary hepatocyte culture (CM0-3) is able to induce prominent changes in cell morphology and reveals a time dependent apoptosis inducing activity in primary cultures of rat hepatocytes. These effects can be modulated by different growth promoting regimens. Notably, apoptosis induction can be inhibited by proliferative stimulation or treatment with dexamethasone, a synthetic corticosteroid which is known to stabilize differentiated hepatocyte functions. In addition, CM0-3 also induces apoptosis in transformed human hepatocytes (human hepatocarcinoma cells) even under culture conditions, which reduce apoptosis induction in cultures of primary (= non-transformed) rat hepatocyte cultures.

Since well known apoptosis inducers in hepatocytes (TGFβ , TNFα and FasL) could be excluded as candidates responsible for CM0-3 mediated apoptosis purified the apoptosis inducing activity. In collaboration with Prof. Lottspeich, MPI- Martinsried – Munich ferritin was identified as apoptosis inducer.

Ferritin

Ferritin, a regulator of the intracellular iron homeostasis, is a multimeric protein composed of 24 heavy (H) and light (L) chain subunits comprising tissue specific isoforms with varying ratios of heavy to light chains. Based on its composition, these ferritin isoforms can also be distinguished as basic (L-rich) and acidic (H-rich) isoferritins. Under normal conditions, cells release only small amounts of ferritin into the bloodstream, however, the serum levels of H-chain rich acidic isoferritins are increased during pregnancy and several pathological conditions such as acute lymphocytic leukemia, Hodgkin‘s disease, breast cancer and certain liver diseases.

Hepatocyte derived acidic isoferritins, which share homology with PLF (placental ferritin) and melanoma derived isoferritin, are able to stimulate apoptosis in hepatocytes by addressing the Fas pathway and intrinsic mitochondrial signalling mediated by the bcl-family member Bid.

Since ferritin is well known as an iron storage protein, it can be speculated that an increased release of Fe2+ from the protein leads to the formation of hydroxyl radicals via the Fenton reaction inducing lipid peroxidation. Lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE), are potent mutagens causing DNA damage and protein modification. Interestingly, HNE-modified proteins and micronuclei resulting from DNA damage were found in cultures of primary rat hepatocytes treated with acidic isoferritins. Furthermore HNE modified proteins colocalised with ferritin after immunohistochemical staining. These results clearly point to Fenton reaction driven oxidative stress in ferritin mediated apoptosis.

Growing evidence indicates oxidative stress as a mechanism of several pathological conditions originating from chronic inflammation such as hepatitis and hepatocellular carcinomas (HCC). Interestingly, levels of serum ferritin are markedly increased in hepatocarcinomas and certain liver diseases (hepatitis).

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