Human influence on brown trout juvenile body size during metapopulation expansion.

Change in body size can be driven by social (density) and non-social (environmental and spatial variation) factors. In expanding metapopulations, spatial sorting by means of dispersal on the expansion front can further drive the evolution of body size. However, human intervention can dramatically affect these founder effects. Using long-term monitoring of the colonization of the remote Kerguelen islands by brown trout, a facultative anadromous salmonid, we analyse body size variation in 32 naturally founded and 10 human-introduced populations over 57 years. In naturally founded populations, we find that spatial sorting promotes slow positive changes in body size on the expansion front, then that body size decreases as populations get older and local density increases. This pattern is, however, completely different in human-introduced populations, where body size remains constant or even increases as populations get older. The present findings confirm that changes in body size can be affected by metapopulation expansion, but that human influence, even in very remote environments, can fully alter this process.

Development and validation of an ultra high performance liquid chromatography-electrospray tandem mass spectrometry method using selective derivatisation, for the quantification of two reactive aldehydes produced by lipid peroxidation, HNE (4-hydroxy-2(E)-nonenal) and HHE (4-hydroxy-2(E)-hexenal) in faecal water.

Red or processed meat rich diets have been shown to be associated with an elevated risk of colorectal cancer (CRC). One major hypothesis involves dietary heme iron which induces lipid peroxidation. The quantification of the resulting reactive aldehydes (e.g. HNE and HHE) in the colon lumen is therefore of great concern since these compounds are known for their cytotoxic and genotoxic properties. UHPLC-ESI-MS/MS method has been developed and validated for HNE and HHE quantification in rat faeces. Samples were derivatised using a brominated reagent (BBHA) in presence of pre-synthesized deuterated internal standards (HNE-d11/HHE-d5), extracted by solid phase extraction, and then analysed by LC-positive ESI-MS/MS (MRM) on a TSQ Vantage mass spectrometer. The use of BBHA allowed the efficient stabilisation of the unstable and reactive hydroxy-alkenals HNE and HHE. The MRM method allowed selective detection of HNE and HHE on the basis of characteristic transitions monitored from both the 79 and 81 bromine isotopic peaks. This method was validated according to the European Medicines Agency (EMEA) guidelines, by determining selectivity, sensitivity, linearity, carry-over effect, recovery, matrix effect, repeatability, trueness and intermediate precision. The performance of the method enabled the quantification of HNE and HHE in concentrations 0.10-0.15 µM in faecal water. Results are presented on the application to the quantification of HNE and HHE in different faecal waters obtained from faeces of rats fed diets with various fatty acid compositions thus corresponding to different pro-oxidative features.

Cell death and diseases related to oxidative stress: 4-hydroxynonenal (HNE) in the balance.

During the last three decades, 4-hydroxy-2-nonenal (HNE), a major α,ß-unsaturated aldehyde product of n-6 fatty acid oxidation, has been shown to be involved in a great number of pathologies such as metabolic diseases, neurodegenerative diseases and cancers. These multiple pathologies can be explained by the fact that HNE is a potent modulator of numerous cell processes such as oxidative stress signaling, cell proliferation, transformation or cell death. The main objective of this review is to focus on the different aspects of HNE-induced cell death, with a particular emphasis on apoptosis. HNE is a special apoptotic inducer because of its abilities to form protein adducts and to propagate oxidative stress. It can stimulate intrinsic and extrinsic apoptotic pathways and interact with typical actors such as tumor protein 53, JNK, Fas or mitochondrial regulators. At the same time, due to its oxidant status, it can also induce some cellular defense mechanisms against oxidative stress, thus being involved in its own detoxification. These processes in turn limit the apoptotic potential of HNE. These dualities can imbalance cell fate, either toward cell death or toward survival, depending on the cell type, the metabolic state and the ability to detoxify.

Liquid chromatography/electrospray ionisation mass spectrometric tracking of 4-hydroxy-2(E)-nonenal biotransformations by mouse colon epithelial cells using [1,2-13C2]-4-hydroxy-2(E)-nonenal as stable isotope tracer.

4-Hydroxy-2(E)-nonenal (HNE), a product of lipid peroxidation, has been extensively studied in several areas, including metabolism with radio-isotopes and quantification in various matrices with deuterium-labelled HNE as standard. The aim of this work was to evaluate the relevance of (13)C-labelled HNE in biotransformation studies to discriminate metabolites from endogens by liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS). (13)C-Labelled HNE was synthesised in improved overall yield (20%), with the incorporation of two labels in the molecule. Immortalised mouse colon epithelial cells were incubated with 2:3 molar amounts of HNE/(13)C-HNE in order to gain information on the detection of metabolites in complex media. Our results demonstrated that the stable isotope m/z values determined by mass spectrometry were relevant in distinguishing metabolites from endogens, and that metabolite structures could be deduced. Six conjugate metabolites and 4-hydroxy-2(E)-nonenoic acid were identified, together with an incompletely identified metabolite. Stable-isotope-labelled HNE has already been used for quantification purposes. However, this is the first report on the use of (13)C-labelled HNE as a tracer for in vitro metabolism. (13)C-Labelled HNE could also be of benefit for in vivo studies.

Chemistry and biochemistry of lipid peroxidation products.

Oxidative stress and resulting lipid peroxidation is involved in various and numerous pathological states including inflammation, atherosclerosis, neurodegenerative diseases and cancer. This review is focused on recent advances concerning the formation, metabolism and reactivity towards macromolecules of lipid peroxidation breakdown products, some of which being considered as 'second messengers' of oxidative stress. This review relates also new advances regarding apoptosis induction, survival/proliferation processes and autophagy regulated by 4-hydroxynonenal, a major product of omega-6 fatty acid peroxidation, in relationship with detoxication mechanisms. The use of these lipid peroxidation products as oxidative stress/lipid peroxidation biomarkers is also addressed.

Apc mutation induces resistance of colonic cells to lipoperoxide-triggered apoptosis induced by faecal water from haem-fed rats.

Recent epidemiological studies suggest that high meat intake is associated with promotion of colon cancer linked with haem-iron intake. We previously reported that dietary haem, in the form of either haemoglobin or meat, promotes precancerous lesions in the colon of rats given a low-calcium diet. The mechanism of promotion by haem is not known, but is associated with increased lipid peroxidation in faecal water and strong cytotoxic activity of faecal water on a cancerous mouse colonic epithelial cell line. To better understand the involvement of faecal water components of haem-fed rats in colon-cancer promotion, we explored the effect of faecal water on normal [adenomatous polyposis coli (Apc)+/+] or premalignant cells (Apc-/+). Further, we tested if this effect was correlated to lipoperoxidation and 4-hydroxynonenal (HNE). We show here for the first time that heterozygote Apc mutation represents a strong selective advantage, via resistance to apoptosis induction (caspase 3 pathway), for colonic cells exposed to a haem-iron-induced lipoperoxidation. The fact that HNE treatment of the cells provoked the same effects as the faecal water of rats fed the haem-rich diet suggests that this compound triggers apoptosis in those cells. We propose that this mechanism could be involved in the promotion of colon carcinogenesis by haem in vivo.

Liquid chromatography-multistage tandem mass spectrometry for the quantification of dihydroxynonene mercapturic acid (DHN-MA), a urinary end-metabolite of 4-hydroxynonenal.

The mercapturic acid conjugate of 1,4-dihydroxynonene (DHN-MA) is a urinary metabolite of 4-hydroxynonenal (4-HNE), one of the main lipid peroxidation products occurring in vivo. To determine its level in urine, a combination of liquid chromatography with positive electrospray-multistage tandem mass spectrometry has been developed. A deuterated analog of the target compound (DHN-MA) with six deuterium atoms was synthesized and used as the internal standard. Three-stage tandem mass spectrometry was used, providing good selectivity for the detection of DHN-MA. The response of the system to DHN-MA was linear in the 5-100 ng range. Urine samples spiked with different levels of standard DHN-MA were used to evaluate the influence of matrix effects on the linearity. The repeatability of the method was also determined by using repeated 5 ng injections of DHN-MA, providing a RSD of 10%. The method was then applied to the determination of DHN-MA in rat urine samples; increased levels of urinary DHN-MA in urine from rats treated with BrCCl3 indicates that lipid peroxidation processes take place in such rats.

Uterotrophic effect of a saturated fatty acid 17-ester of estradiol-17beta administered orally to juvenile rats.

In comparison to estradiol-17beta, the naturally synthesized estradiol-17beta-17-fatty acid esters are potent estrogens when administered subcutaneously. A lipophilic character of estradiol-17-esters could partially protect them from metabolic inactivation. In order to compare their relative estrogenic potency when administered orally, the uterotrophic response to different dosages (0, 2.5, 25, 250 and 2500 nmol/kg BW/day) of estradiol-17beta and estradiol-17beta-17-stearate was assessed in juvenile Sprague-Dawley female rats. Estrogens were administered by oral gavage once a day for 6 days. On the 7th day uterus and vagina were dissected, weighed, and examined microscopically. At 2.5 and 25 nmol/kg BW/day, no difference was detected in the uterus weight compared to control animals which received the vehicle alone (corn oil). At 250 nmol/kg BW/day, the uterotrophic response was maximal in estradiol-17beta-17-stearate-treated animals (x2.40-2.70), whereas it was moderate in estradiol-17beta-treated rats (x1.86) at the same dosage. This differential weight gain effect of estradiol-17beta-17-stearate was correlated with typical microscopic changes in uterus and vagina. The results are in favour of a stronger estrogenic effect of orally given lipoidal estrogens compared to estradiol-17beta. This could be explained by a slower but sustained absorption of estradiol-17beta released from estradiol-17beta-17-stearate by esterases and/or by a facilitated transfer of esters in the lymphatic circulation.

In vivo involvement of cytochrome P450 4A family in the oxidative metabolism of the lipid peroxidation product trans-4-hydroxy-2-nonenal, using PPARalpha-deficient mice.

Trans-4-hydroxy-2-nonenal (HNE) is a potent cytotoxic and genotoxic compound originating from the peroxidation of n-6 polyunsaturated fatty acids. Its metabolism has been previously studied in the rat (Alary et al. 1995. Chem. Res. Toxicol., 8: 35-39). In addition to major urinary mercapturic derivatives, some polar urinary metabolites were isolated and could correspond to hydroxylated compounds. 4-Hydroxynonenoic acid (HNA), resulting from the oxidation of the HNE carbonyl group, is a medium chain fatty acid and its omega-hydroxylation might be hypothesized. Therefore, the involvement of the CYP 4A family isoenzymes in the metabolism of [3H]HNE has been investigated in vivo using inducer treatments (fibrates) in wild-type or in peroxisome proliferator-activated receptor alpha (PPARalpha)-deficient mice. In wild-type mice, but not in PPARalpha (-/-) mice, fibrate treatments resulted in an increase of two urinary metabolites characterized, after HPLC purifications and mass spectrometry analyses, as the omega-hydroxylated metabolite of HNA, i.e., 4,9-dihydroxy-2-nonenoic acid, and its oxidized form, 4-hydroxy-2-nonene-1,9-dicarboxylic acid. The formation of the latter is correlated accurately to laurate hydroxylase activity studied concurrently in microsomes prepared from the liver of these animals. Basal levels of these two metabolites were measured in urine of normal and PPARalpha-deficient mice. These results are in accord with an implication of the P450 4A family in the extended oxidative metabolism of 4-HNE.

Glucuronidation: a dual control.

1. Glucuronidation is a major detoxication process catalyzed by uridine diphosphate glucuronosyltransferases. 2. The amount of enzyme can be modulated by numerous foreign compounds, such as common chemical inducers already implicated in the induction of other detoxication enzymes. 3. Hormones such as thyroid hormones or growth hormone also are implicated in the control of glucuronidation. 4. Because glucuronidation enzymes (isozymes) are anchored in the endoplasmic reticulum membrane, with their active site likely being located on the lumenal side of the membrane, the membrane environment of these enzymes was shown to modulate their functional state as evaluated by the conjugating activity per enzymatic molecular unit. 5. In accord with a first, previously proposed model, it seems that this modulation can be attributed to different conformational states of the enzymes, depending on the physicochemical state of the membrane. 6. In accord with a second model, the membrane may act as a barrier between the enzymes and the cosubstrate UDP-glucuronic acid, which is a polar and charged molecule synthesized in the cytosol. This would imply a transporting process for this molecule through the reticulum membrane, which has been characterized in vitro and could be of importance in vivo. 7. Glucuronidation is under the control of a dual regulation, by means of a specific isozyme expression level and by the modulation of their functional state.

Hepatic microsomal membrane lipidic composition and growth hormone effect in adult male rat: evidence for a 'feminization' process of total phospholipid fatty acid pattern.

Growth hormone (GH) effects on fatty acid composition and on delta5-, delta6-, delta9-desaturase and palmitic acid elongation activities were studied in male rat hepatic microsomes. Sham-operated and hypophysectomized animals were injected with two different dosages of GH, mimicking either the male or female GH secretion pattern. Half the hypophysectomized animals received thyroxine and cortisol in concentrations chosen to compensate for the lack of thyroid hormones and glucocorticoids. GH, administered to sham-operated or to cortisol/thyroxine-treated hypophysectomized rats resulted in an increase in stearic and arachidonic acid proportions, while palmitic acid percentage was decreased. Total monounsaturated fatty acids were dramatically reduced by this treatment. DeltaA-desaturase and palmitic acid elongation activities were increased by GH treatment, while delta9-desaturase activity was decreased. These GH effects on desaturation and elongation activities could explain the modifications in microsomal fatty acid composition. Hypophysectomy markedly altered the fatty acid composition by reducing arachidonic and stearic acid proportions and increasing the linoleic acid proportion, while delta9-, delta5-desaturase and palmitic acid elongation activities were decreased. Restoration of most of the fatty acid proportions to control values was realized in hypophysectomized animals with a cortisol/thyroxine replacement administered alone or together with the low dosage of GH mimicking the male secretion pattern. High GH dosage produces essentially a 'feminization' process of the fatty acid composition of the hepatic microsomal membrane in male rats when compared to that of females.

Differential effect of hypophysectomy and growth hormone treatment on hepatic glucuronosyltransferases in male rats: evidence for an action at a pretranslational level for isoforms glucuronidating bilirubin.

The influence of growth hormone (GH) on 4-nitrophenol, bilirubin, testosterone, androsterone and estrone glucuronidation activities was studied in fully activated male rat hepatic microsomes. Sham-operated and hypophysectomized animals were injected with two different dosages of GH, mimicking either the male or female GH secretion pattern. Half the animals received thyroxine and cortisol in concentrations chosen to compensate for the lack of thyroid hormones and glucocorticoids in hypophysectomized rats. GH induced a decrease in several glucuronidation activities: bilirubin glucuronidation in both sham-operated and cortisol/ thyroxine-treated hypophysectomized rats in a dose-dependent manner, testosterone glucuronidation in hypophysectomized animals, and androsterone and estrone glucuronidation in cortisol/thyroxin-treated hypophysectomized rats. 4-nitrophenol glucuronidation was not affected by GH treatment. A hypothetical "feminizing" effect of GH (due to an almost continuous secretion) could not be invoked to explain these results, contrary to what has been observed elsewhere for other hepatic enzyme activities. Hypophysectomy altered all the activities tested, with bilirubin the most modified (a 200% enhancement). Restoration of control values was achieved in hypophysectomized animals with cortisol/thyroxine replacement together with a low dosage of GH (mimicking a male GH secretion pattern), except for androsterone glucuronidation activity where both GH and cortisol/thyroxine treatments reinforced the decreasing effect of hypophysectomy. Variations in protein amounts were correlated to variations in bilirubin, testosterone and androsterone conjugation activities induced by hypophysectomy and GH treatment. Reverse transcription-polymerase chain reaction (RT-PCR) mRNA analysis of bilirubin cluster isoforms or uridine diphosphate glucuronosyltransferase 1B1 (UGT1B1), UGT1B2 and UGT1B5 showed that GH controlled the different isoforms involved in bilirubin glucuronidation differentially at a pretranslational level.