Formation of 4-hydroxynonenal and further aldehydic mediators of inflammation during bromotrichlorornethane treatment of rat liver cells

Bromotrichloromethane (CBrCl3) treatment is a model for studies on molecular mechanisms of haloalkane toxicity with some advantages compared with CCl4 treatment. The formation of 4-hydroxynonenal and similar aldehydic products of lipid peroxidation, which play a role as mediators of inflammatory processes, was clearly demonstrated in rat hepatocytes treated with CBrCl3. It may be assumed that haloalkane toxicity is connected with the biological effects of those inflammation mediatory aldehydic compounds.


Introduction
Although pathophysiological and biochemical changes due to haloalkane intoxication have been investigated intensively by many laboratories, increasing exposure to toxic pollutants in the workplace and in the environment requires the continuation of studies on the molecular basis of haloalkane toxicity. Haloalkanes are also of importance in clinical practice because of the use of halothane and other halogenated compounds as inhalation anaesthetics, and a number of clinical cases of CC14 poisoning. Additionally 1,2-dibromomethane has been used as a fruit and grain fumigant.
Haloalkanes induce serious inflammatory reactions. Several reports exist on biochemical changes in plasma during halothane treatment characterizing these inflammatory reactions, 1'2 and on hepatitis and cirrhosis after halothane or CC14 intoxication. The cytotoxic mechanisms of haloalkanes have been studied mostly in cell suspensions, organs or whole animals treated with CC14. CBrC13 was also used several times for investigations on haloalkane toxicity. CC14 is metabolized in a cytochrome P-450 dependent reaction which leads to the formation of the "CC13 radical. The "CC13 radical rapidly interacts with membrane lipids. [4][5][6][7] The "CC13 induced lipid peroxidation following CC14 treatment is con-siderably lower than that induced by CBrC13 treatment. It is known that the C-C1 cleavage needs more energy (68 kcal/mol) than the cleavage of the C-Br bond (49 kcal/mol). Koch

Results
In this study of HNE,, MDA and further aldehydic products of lipid peroxidation could be detected and quantitatively evaluated in the samples of hepatocyte suspensions. There were no differences between HNE levels of controls and CBrC13

Discussion
Pathophysiological role of aldehydic products of lipid peroxidation: There is increasing evidence that aldehydes generated during the process of lipid peroxidation are causally involved in some of the pathophysiological effects associated with oxidative stress in cells and tissues. 2 Unlike reactive free radicals, aldehydes are rather long-lived and can therefore diffuse from the site of their origin, i.e. membranes, and reach and attack targets intracellularly or extracellularly which are distant from the initial free radical event. HNE and similar aldehydic products of lipid peroxidation exhibit a wide spectrum of cytotoxic effects as well as mutagenic and genotoxic effects, e-23 Since HNE, and other aldehydes, are formed during the acute phase of inflammation they may play a role as mediators in the inflammatory process. 2 HNE effects produced even at HNE concentrations of 0.1/M or less include stimulation of chemotactic oriented migration of neutrophils, 12'24'2s modulation of adenylate cyclase activity, weak stimulation of guanylate cyclase 26 and stimulation of phospholipase C. iv Phospholipase C is important for signal transduction by G-proteins. Stimulation of phospholipase C leads to increased hydrolysis of phosphatidyl-4,5-bisphosphate with concomitant formation of diacylglycerol and inositol triphosphate. Diacylglycerol acts synergistically with inositol triphosphate in the calcium dependent activation of protein kinase C. Inositol triphosphate opens calcium channels, whereas diacylglycerol increases the calcium affinity of protein kinase C. Protein kinase C plays an important role in the control of cell division, cell proliferation and other cellular functions. Phospholipase C stimulation may also be responsible for the chemotactic effects of 4-hydroxynonenal since it is known that the chemotactic signal of f-met peptides also results in an activation of phospholipase. 28 4-Hydroxyalkenals induce the oriented migration and morphological polarization of neutrophils in a concentration range of 10 -6 to 10-12 M depending on the chain length of the aldehyde. The most effective aldehyde was not HNE but 4-hydroxyoctenal with a maximum chemotactic activity at 10 -1 M. For HNE the effective doses for chemotactic effects and for activation of phospholipase C are between 10 -6 and 10 -9 M. 2s Cytotoxic haloalkane effects depend on the formation and action of free radicals initiating lipid peroxidation. 4'5'29 The formation of HNE and similar aldehydic products of lipid peroxidation in hepatocytes treated with CC14 was published by Poli et al. In the present study the formation of such compounds could also be demonstrated for CBrCI. 30 Mediators of Inflammation. Vol 2.1993 Critical aspects of methodological approach: For the determination of long chain aldehydes a number Of methods, such as the dinitrophenylhydrazine method, the direct determination by HPLC, the cyclohexadione method and the determination by GC-MS, are described in Esterbauer and Zollner. The determination of a wide spectrum of aliphatic aldehydes is only possible, using the dinitrophenylhydrazine and the cyclohexadione method, with comparable results in recovery and reproducibility. The literature results for reproducibility and low recovery (about 40%) are in accordance with those achieved in this study. ' The determination of the relatively low recoveries for each aldehyde allows the determination of the real biological steady state concentration in the cell suspension. Nevertheless, the low recovery and therefore a certain inaccuracy of methods which were available for aldehyde analysis modify the quantitative aspect of those measurements.
Compar#on of formation of aldehydes as mediators of inflammation in CC14 and CBrC13 toxicity: The initial levels of MDA and of HNE at zero time were in the same range as concentrations reported by other authors in hepatocyte suspensions. 13 In contrast to CC14 treated cells the aldehyde levels increased very rapidly, i.e. within the first minutes following CBrCI addition to the hepatocytes. However, the increases of MDA and HNE levels in hepatocyte suspensions treated with CBrCI were in the same range as in those hepatocyte suspensions treated with equimolar concentrations of CC14 .13 The increase of some aldehydic products of lipid peroxidation within 1 h of treatment with CC14 or CBrCI in comparison to initial levels is demonstrated in Fig. 3. The most interesting point of this comparison seems to be the excessive increase of HNE as a very cytotoxic and chemotactic compound at CBrCI loading compared with CC14 loading.
The generation of aldehydic products of lipid peroxidation other than MDA and HNE could be demonstrated for CBrC13 incubations (Table 1) but at markedly lower concentrations than those for MDA and HNE. Nevertheless, the amounts of these aldehydes and of HNE which were produced even during short-term experiments are high enough for the initiation of chemotactic activity towards neutrophils.
It should be mentioned that the determined aldehyde concentrations represent, of course, the steady state concentrations, resulting from a continuous formation of the aldehydes and a consumption of these compounds by the hepatocytes. The HNE metabolism--including that in hepatocytes--was investigated in several studies. 1 '2-5 In general the maximal capacity of tissues to metabolize aldehydes, especially HNE, is in the order of several micromoles per gram wet weight per minute, which is about six orders higher than the accumulation within the first minutes after haloalkane treatment.
The specific pattern of aldehydes formed by CBrC13 and CC14 is different, whereas the overall increase in MDA and HNE is comparable for both compounds. The differences in lifetime, reactivity and 'consumption' rate of aldehydes may implicate differences in the pattern of cell and tissue damage if a different aldehyde pattern occurs. There is the possibility that in CBrC13 induced liver damage 4-hydroxynonenal and similar aldehydic products of lipid peroxidation are involved in cell damage and act as mediators of inflammatory processes. The relation between formation and 'consumption' of aldehydes should be investigated in further experiments.