Toxic effects of microbial phenolic acids on the functions of mitochondria

Abstract

Low-molecular-weight phenolic acids (PhAs) phenylacetate, phenyllactate, phenylpropionаte, р-hydroxyphenyllactate, and p-hydroxyphenylacetate are essentially the products of the degradation of aromatic amino acids and polyphenols by the intestinal microflora. In sepsis, the concentrations of some of these acids in the blood increase tens of times. Assuming that these compounds can cause the mitochondrial dysfunction in sepsis, we examined their effects on respiration, the induction of pore opening, and the production of reactive oxygen species (ROS) in mitochondria. It was found that phenylpropionаte and phenylacetate produce a more toxic effect on mitochondria than the other phenolic acids. At concentrations 0.01–0.1 mM they decreased the rate of oxidation of NAD-dependent substrates and activated the Ca²⁺- and menadione-induced opening of the cyclosporin A-sensitive pore and the production of ROS. The disturbances caused by these PhAs are similar to those observed in mitochondria in sepsis, and hence the rise in their level may be one of the causes of mitochondrial dysfunctions. Phenyllactate, р-hydroxyphenyllactate, and p-hydroxyphenylacetate inhibited the production of ROS and pore opening, acting as antioxidants. Thus, the ability of PhAs to affect the mitochondrial functions, as well as an increase in their concentrations in sepsis (the total concentration of these PhAs in the blood is close to 0.1 mM), suggests that PhAs can be directly involved in the development of mitochondrial failure.

Introduction

Phenolic acids (PhAs) are formed in vivo in several metabolic pathways. They are the products of oxidation of aromatic amino acids in pathologies associated with the deficit of the enzyme phenylalanine hydroxylase, which catalyzes the formation of tyrosine from phenylalanine (Deutsch, 1997, Sarkissian et al., 2000, Antoshechkin et al., 1991). These pathologies are accompanied by elevated levels of phenylacetate, phenyllactate, and phenylpyruvate in body fluids. Phenolic compounds are also found in body fluids in norm. In this case, they arise from the anaerobic degradation of aromatic amino acids by the intestinal microflora. According to the data reported in the literature, the concentrations of some PhAs in the plasma of healthy adults vary from 68 to 226 ng/ml (Deutsch, 1997, Sarkissian et al., 2000, Antoshechkin et al., 1991). In rats, the concentration of endogenous circulating p-hydroxyphenylacetate in plasma is considerably higher, 410 ng/ml or 2.7 μmol/l (Mani et al., 2003).

Another possible way of the formation of low-molecular-weight PhAs is the degradation by the intestinal microflora of dietary polyphenolic compounds. It is known that the chemical structure of polyphenols determines the extent of intestinal absorption and the nature of the their metabolites circulating in the plasma (Aura et al., 2002, Déprez et al., 2000, Scalbert and Williamson, 2000). Low-molecular-weight metabolites of polyphenols are formed by the colonic microflora and not in animal tissues, as indicated by the suppression of their formation after the administration of antibiotics (Scalbert and Williamson, 2000). In humans, 4-hydroxyphenylacetic acid, phenylpropionаte, 4-hydroxyphenylpropionаte, and 3,4-dihydroxyphenylacetic acid in vivo were identified as the metabolites of quercetin, catechin, and other polyphenols (Déprez et al., 2000, Scalbert and Williamson, 2000). The total concentration of phenolic compounds in fecal water samples was 789 ± 66 μmol/l, whereas the concentration range of individual derivatives was 0.01–417 μmol/l. The most abundant phenolic compounds were phenylpropionic and phenylacetic acids, 188 and 197 μmol/l, respectively, whereas the concentrations of individual hydroxyl derivatives were below 100 μmol/l (Jenner et al., 2005, Karlsson et al., 2005).

Low-molecular-weight PhAs can influence cellular functions, such as genotoxicity, proliferation, apoptosis, and cell signaling (Scalbert and Williamson, 2000, Jenner et al., 2005, Li et al., 2004, Karlsson et al., 2005). Some of them affect directly the enzymes. p-Hydroxyphenylacetate is a potent noncompetitive inhibitor of dihydropteridine reductase, with K_i values in the range of 4–260 μM (Shen, 1984). This metabolite can affect the level of tetrahydrobiopterin, a cofactor of tyrosine hydroxylation, thereby disturbing the biosynthesis of 3,4-dihydroxyphenylalanine and adrenaline. An important feature of many PhAs is their effect on the activity of cyclooxygenase, which plays a major role in the regulation of inflammation. 3-Phenylpropionic and 3-hydroxyphenylacetic acids at concentrations of 250 μM and above decreased both the protein level and the enzymatic activity of the inducible enzyme cyclooxygenase-2 (Karlsson et al., 2005). In cell cultures, low-molecular-weight PhAs can produce both the cytoprotective and cytotoxic effects. It was found on hepatocytes that gallic acid as a model of a low-molecular-weight PhAs produces the cytotoxic action, which is mediated through the activation of the formation of mitochondrial reactive oxygen species (ROS) (Galati et al., 2006). It is also known that hydroxylated PhAs are the substrates of peroxidase and are oxidized in the presence of hydrogen peroxide to phenolic acid dimers (Panus et al., 1993, Genfa and Dasgupta, 1992).

Previously, we have shown that the content of some PhAs in the blood increases substantially in sepsis (Beloborodova et al., 2006, Khodakova and Beloborodova, 2007). The most pronounced changes were observed for р-hydroxyphenyllactate; its level increased almost by two orders of magnitude, from 0.47 to 33.2 μM. The concentrations of phenylacetate, phenyllactate, 3-phenylpropionаte, and p-hydroxyphenylacetate varied from 0.68 μM in norm to 14.3 μM in sepsis. These compounds are produced by clinically important bacteria, such as K. pneumonia > E. coli > S. aureus > S. marcesceus, E. cloacae, and S. epidermidis. Assuming that these compounds can contribute to the mitochondrial dysfunction developing in sepsis, we examined their effect on respiration, the induction of pore opening, and the production of ROS in mitochondria. We showed that PhAs at high concentrations (0.2–0.5 mM) decrease the rate of oxidation of NAD-dependent substrates and have no effect on the oxidation of succinate. At considerably lower concentrations (0.01–0.1 mM), they affect the opening of the Ca-dependent, cyclosporin (Cs) A-sensitive pore and the production of ROS. The effect of PhAs manifests itself either in the activation or the inhibition of pore opening and ROS production, depending on the structure of the compound.