Genotoxicity of acrylamide in vitro: Acrylamide is not metabolically activated in standard in vitro systems

The recent finding that acrylamide (AA), a genotoxic rodent carcinogen, is formed during the frying or baking of a variety of foods raises human health concerns. AA is known to be metabolized by cytochrome P450 2E1 (CYP2E1) to glycidamide (GA), which is responsible for AA's in vivo genotoxicity and probable carcinogenicity. In in‐vitro mammalian cell tests, however, AA genotoxicity is not enhanced by rat liver S9 or a human liver microsomal fraction. In an attempt to demonstrate the in vitro expression of AA genotoxicity, we employed Salmonella strains and human cell lines that overexpress human CYP2E1. In the umu test, however, AA was not genotoxic in the CYP2E1‐expressing Salmonella strain or its parental strain. Moreover, a transgenic human lymphoblastoid cell line overexpressing CYP2E1 (h2E1v2) and its parental cell line (AHH‐1) both showed equally weak cytotoxic and genotoxic responses to high (>1 mM) AA concentrations. The DNA adduct N7‐GA‐Gua, which is detected in liver following AA treatment in vivo, was not substantially formed in the in vitro system. These results indicate that AA was not metabolically activated to GA in vitro. Thus, AA is not relevantly genotoxic in vitro, although its in vivo genotoxicity was clearly demonstrated. Environ. Mol. Mutagen. 52:11–19, 2011. © 2010 Wiley‐Liss, Inc.     

Genotoxicity of acrylamide and its metabolite glycidamide administered in drinking water to male and female Big Blue mice

The recent discovery of acrylamide (AA), a probable human carcinogen, in a variety of fried and baked starchy foods has drawn attention to its genotoxicity and carcinogenicity. Evidence suggests that glycidamide (GA), the epoxide metabolite of AA, is responsible for the genotoxic effects of AA. To investigate the in vivo genotoxicity of AA, groups of male and female Big Blue (BB) mice were administered 0, 100, or 500 mg/l of AA or equimolar doses of GA, in drinking water, for 3-4 weeks. Micronucleated reticulocytes (MN-RETs) were assessed in peripheral blood within 24 hr of the last treatment, and lymphocyte Hprt and liver cII mutagenesis assays were conducted 21 days following the last treatment. Further, the types of cII mutations induced by AA and GA in the liver were determined by sequence analysis. The frequency of MN-RETs was increased 1.7-3.3-fold in males treated with the high doses of AA and GA (P < or = 0.05; control frequency = 0.28%). Both doses of AA and GA produced increased lymphocyte Hprt mutant frequencies (MFs), with the high doses producing responses 16-25-fold higher than that of the respective control (P < or = 0.01; control MFs = 1.5 +/- 0.3 x 10(-6) and 2.2 +/- 0.5 x 10(-6) in females and males, respectively). Also, the high doses of AA and GA produced significant 2-2.5-fold increases in liver cII MFs (P < or = 0.05; control MFs = 26.5 +/- 3.1 x 10(-6) and 28.4 +/- 4.5 x 10(-6)). Molecular analysis of the mutants indicated that AA and GA produced similar mutation spectra and that these spectra were significantly different from that of control mutants (P < or = 0.001). The predominant types of mutations in the liver cII gene from AA- and GA-treated mice were G:C-->T:A transversions and -1/+1 frameshifts in a homopolymeric run of Gs. The results indicate that both AA and GA are genotoxic in mice. The MFs and types of mutations induced by AA and GA in the liver are consistent with AA exerting its genotoxicity in BB mice via metabolism to GA.     

Renal liver-type fatty acid binding protein (L-FABP) attenuates acute kidney injury in aristolochic acid nephrotoxicity

Injection of aristolochic acid (AA) in mice causes AA-induced nephrotoxicity, in which oxidative stress contributes to development of tubulointerstitial damage (TID). Liver-type fatty acid binding protein (L-FABP) is expressed in human proximal tubules and has an endogenous antioxidative function. The renoprotection of renal L-FABP was examined in a model of AA-induced nephrotoxicity. Established human L-FABP (hL-FABP) transgenic (Tg) mice and wild-type (WT) mice were treated with AA for up to 5 days. Mice were sacrificed on days 1, 3, and 5 after the start of AA injection. Although mouse L-FABP was not expressed in proximal tubules of WT mice, hL-FABP was expressed in proximal tubules of Tg mice. The expression of renal hL-FABP was significantly increased in Tg mice administered AA (Tg-AA), compared with the control (saline-treated Tg mice). In WT-AA mice, there was high urinary excretion of N(ε)-(hexanoyl)-lysine, the production of heme oxygenase-1 and receptor for advanced glycation end products increased, and TID was provoked. In contrast, renal hL-FABP in Tg-AA mice suppressed production of N(ε)-(hexanoyl)lysine, heme oxygenase-1, and receptor for advanced glycation end products. Renal dysfunction was significantly milder in Tg-AA mice than in WT-AA mice. The degree of TID was significantly attenuated in Tg-AA mice, compared with WT-AA. In conclusion, renal hL-FABP reduced the oxidative stress in AA-induced nephrotoxicity and attenuated TID.     

Sex-differential modulation of visceral pain by brain derived neurotrophic factor (BDNF) in rats

In spite of the fact that brain derived neurotrophic factor (BDNF) has been reported to be implicated in the development of visceral pain, it remains to be determined whether the role of BDNF in pain is gender dependent. The present study investigated the effect of BDNF on visceral pain in different gender rats. A model for visceral pain was established by intraperitoneal (i.p.) injection of acetic acid (AA) into Sprague–Dawley rats: males, females and females with an ovariectomy (OVX). The pain behavior index was assessed by counting the number of abdominal contractions for 60 min after i.p. injection of AA. Anti-BDNF antibody, or BDNF, was administered 1 h before the AA injection to examine the role of BDNF in visceral pain. After the AA injection, the number of abdominal contraction was dramatically increased in all rats but females showed more severe pain behavior than males. The higher sensitivity to AA-induced nocifensive response was attenuated by OVX. Pretreatment with anti-BDNF antibody significantly exacerbated the nocifensive response in males but attenuated it in females. While exogenous BDNF administration did not alter AA injection-induced nocifensive response in females, BDNF pretreatment attenuated the nocifensive response in males but exacerbated it in females with OVX. The present study suggests there is a gender dichotomy in visceral pain induced by AA injection. In addition, the modulation of visceral pain by BDNF is also sex dependent, i.e., BDNF facilitates the visceral pain in female rats but displays an opposite effect in male rats. Our results may have important implications in the management of clinical pain.     

Free radical generation and oxidative stress with ageing and exercise: differential effects in the myocardium and liver

Reactive oxygen species and other oxidants are implicated in the mechanisms of biological ageing and exercise-induced tissue damage. The present study examined the effects of ageing and an acute bout of exercise on intracellular oxidant generation, lipid peroxidation, protein oxidation and glutathione (GSH) status in the heart and liver of young adult (8 month, N=24) and old (24 month, N=24) male Fischer 344 rats. Young rats ran on treadmill at 25 m min-1, 5% grade until exhaustion (55.4+/-2.7 min), whereas old rats ran at 15 m min-1, 5% until exhaustion (58.0+/-2.7 min). Rate of dichlorofluorescin (DCFH) oxidation, an indication of intracellular oxidant production, was significantly higher in the homogenates of aged heart and liver compared with their young counterparts. In the isolated heart and liver mitochondria, ageing increased oxidant production by 29 and 32% (P<0.05), respectively. Acute exercise increased oxidant production in the aged heart but not in the liver. When nicodinamide dinucleotide phosphate (reduced), adenosine diphosphate and Fe3+ were included in the assay, DCFH oxidation rate was 47 and 34% higher (P<0.05) in the aged heart and liver homogenates, respectively, than the young ones. The age differences in the induced state reached 83 and 140% (P<0.01) in isolated heart and liver mitochondria, respectively. Lipid peroxidation was increased in the aged liver and exercised aged heart, whereas protein carbonyl content was elevated only in the aged heart (P<0.05). Although our data using DCFH method probably underestimated cellular oxidant production because of time delay and antioxidant competition, it is clear that oxidative stress was enhanced in both heart and liver with old age. Furthermore, aged myocardium showed greater susceptibility to oxidative stress after heavy exercise.     

Aristolochic acid-induced carcinogenesis examined by ACB-PCR quantification of H-Ras and K-Ras mutant fraction

Aristolochic acid (AA) is a strong cytotoxic nephrotoxin and carcinogen associated with the development of urothelial cancer in humans. AA induces forestomach, kidney and urothelial tract tumours in rats and mice. This study was conducted to characterise AA's carcinogenic mechanism of action and compare allele-specific competitive blocker-polymerase chain reaction (ACB-PCR)-based early detection of carcinogenic effect using two different tumour-relevant endpoints. H-Ras codon 61 CAA→CTA mutation was analysed because it is found in rodent forestomach tumours and A:T→T:A transversion is the predominant mutational specificity induced by AA. K-Ras codon 12 GGT→GAT mutation was analysed because it is a common spontaneous mutation present in various rodent tissues and may be a useful generic biomarker for carcinogenic effect. DNA samples from Big Blue rats treated with 0, 0.1, 1.0 or 10.0 mg AA/kg body weight (bw) by gavage, 5 days/week for 12 weeks were used in ACB-PCR in order to examine the induction of the two specific mutations. A significant dose-dependent induction of H-Ras mutant fraction (MF) was observed in liver and kidney. Statistically significant correlations were observed between AA-induced DNA adduct levels or cII mutant frequencies (previously measured in the same rats) and H-Ras MF measurements. No correlation between AA dose and K-Ras MF was found in liver or kidney, although there was a significant induction of K-Ras mutation in kidneys exposed to 0.1 mg/kg bw AA relative to controls. Thus, the data establish a straightforward dose-related increase in H-Ras MF due to fixation of AA-induced DNA adducts, whereas the common spontaneous K-Ras mutation showed a non-monotonic dose-response, consistent with loss of non-targeted mutation at cytotoxic doses.     

Renal vasoconstriction induced by arachidonic acid during burn shock in rats

Metabolites of arachidonic acid are possible mediators of local renal vasoconstriction in burn shock. The prostanoid precursor arachidonic acid (AA) therefore was infused in the control period and at 1 and 2 h after scalding in anaesthetized rats. To avoid systemic effects. AA was infused at low doses directly into the renal artery through a thin cannula introduced through the aortic wall. After control observations 40% of the body surface was scalded in 80 degrees C water. Renal arterial blood flow (RBF), measured by an electromagnetic probe, fell to 70% and 58% of the control level at 1 h and 2 h after scalding respectively. Arterial blood pressure was almost maintained. Infusion of AA (5, 15 and 25 nmol) in the renal artery over 15 s caused no effects in the control period, whereas a dose-dependent decrease in RBF was observed after scalding, and was most pronounced 2 h post-burn. The highest dose of AA reduced RBF by 37% at 1 h and by 80% of preinfusion flow at 2 h after scalding. The AA-induced decrease in RBF was abolished by blocking the thromboxane A2 receptors with AH23848. In contrast, inhibition of prostacyclin synthesis or blocking of serotonin S2 receptors did not significantly influence the response to AA during shock. Thus, infusion of AA into the renal artery caused a marked reduction of RBF after scalding, at doses that did not induce a change during the control period. The augmented effect of AA infusion may be due to an increased capacity for synthesis of thromboxane A2 (TxA2), and possibly PGH2 and PGF2 alpha, after scalding.     

Risk factors of hepatitis C virus-related liver cirrhosis in young adults: positive family history of liver disease and transporter associated with antigen processing 2(TAP2)*0201 Allele

The aim of this study was to clinically characterize young patients with hepatitis-C-related cirrhosis. We compared 27 patients with liver cirrhosis (Group LC) who were anti-HCV positive, aged 40 years or less at the time of diagnosis, with 323 consecutive patients with HCV-related chronic hepatitis (Group CH) matched for age and gender. Furthermore, Group LC was divided into two arbitrary groups (29-35 years, n = 8 /36-40 years, n = 19), based on the age of patients at the time of diagnosis of liver cirrhosis. Patients' characteristics and family history were investigated, and the frequency of transporter associated with antigen processing 2 (TAP2) was determined. A family history of liver disease was present in 40.7% of Group LC but in 18.0% of Group CH (P < 0.05). The younger the age of diagnosis of cirrhosis in Group LC, the higher the frequency of a positive family history (29-35 years, 87.5%; 36-40 years, 21.1%, P < 0.05). The frequency of TAP2*0201 was significantly higher in young adult patients with HCV-related liver cirrhosis than in HCV carriers with normal ALT (P < 0.05), and tended to be higher than in uninfected normal subjects (P = 0.05). The cumulative survival rate of cirrhosis patients with family history of liver diseases was significantly lower than that of cirrhosis patients without such history (P < 0.05). Our findings suggest that a positive family history of liver disease and TAP2*0201 polymorphism may be risk factors for HCV-related liver cirrhosis in young adults.     

Induced development of proximal tubular NaKATPase,basolateral cell membranes and fluid reabsorption

We have previously demonstrated that adrenal corticoid hormones (ACH) induce NaKATPase activity in immature proximal tubular (PT) cells in rats. We have now determined the effect of betamethasone (beta) and aldosterone (aldo) on the size of PT basal and lateral cell membranes (BLM), PT fluid reabsorption (J v (a)) measured in vivo with the split drop technique and PT NaKATPase activity in 20-day-old (young) and 40-day-old (adult) rats. Serum levels of ACH were the same in young and in adult rats, but adrenalectomy caused a significantly larger fall in NaKATPase activity in young than in adult rats. BLM surface area (μm²/μm^-3 cell volume), J v (a) and NaKATPase were significantly lower in young than in adult rats. Three-day treatment with high doses of beta (60 μg· 100 g^-1 or aldo (40 μg· 100 g^-1) significantly increased BLM surface area, J v (a) and NaKATPase activity in young but not in adult rats. Three days after adrenalectomy, J v (a) was significantly depressed in adult rats. There was a significant correlation between the ACH-dependent changes in NaKATPase activity and J v (a). Short-term treatment (2–3 hours) with high doses of ACH did not significantly increase NaKATPase activity and J v (a) in young rats.     

In vivo sister chromatid exchange and micronucleus induction studies with 1,3-butadiene in B6C3F1 mice and Sprague-Dawley rats

Male B6C3F1 mice and Sprague-Dawley rats were exposed for 2 days, 6 h/day to 1,3-butadiene (BD) by inhalation (nose only) and their bone marrow cells were evaluated for the induction of micronuclei (MN) and sister chromatid exchanges (SCEs). A significant dose-dependent increase in MN induction was observed in mice. At 100 p.p.m., the frequency of micronucleated polychromatic erythrocytes was 6-fold above control with a maximal induction of 38-fold at 10,000 p.p.m. A significant increase in SCEs was also observed in mouse bone marrow cells starting at 100 p.p.m. with a 4-fold increase over the control evident at 10,000 p.p.m. The highest tested no observed effect level for both endpoints was 50 p.p.m. In contrast, rat bone marrow cells did not exhibit significant increases in micronucleated polychromatic erythrocytes or SCEs. These results indicate that BD is genotoxic in the bone marrow of the mouse but not the rat. This paralleled the chronic bioassays which showed mice to be more susceptible than rats to BD carcinogenicity.     

Investigation of the genotoxic effect of microwave irradiation in rat bone marrow cells: in vivo exposure

An in vivo mammalian cytogenetic test (the erythrocyte micronucleus assay) was used to investigate the extent of genetic damage in bone marrow red cells of rats exposed to radiofrequency/microwave (RF/MW) radiation. Wistar rats (n = 40) were exposed to a 2.45 GHz continuous RF/MW field for 2 h daily, 7 days a week, at a power density of 5-10 mW/cm(2). The whole body average specific absorption rate (SARs) was calculated to be 1.25 +/- 0.36 (SE) W/kg. Four subgroups were irradiated for 4, 16, 30 and 60 h. Sham-exposed controls (n = 24) were included in the study. The animals of each treated subgroup were killed on the final day of irradiation. Bone marrow smears were examined to determine the extent of genotoxicity after particular treatment times. The results were statistically evaluated using non-parametric Mann-Whitney and Kruskal-Wallis tests. In comparison with the sham-exposed subgroups, the findings of polychromatic erythrocytes (PCE) revealed significant differences (P < 0.05) for experimental days 8 and 15. The frequency of micronucleated PCEs was also significantly increased on experimental day 15 (P < 0.05). Pair-wise comparison of data obtained after 2, 8 and 30 irradiation treatments did not reveal statistically significant differences between sham-exposed and treated subgroups. Under the applied experimental conditions the findings revealed a transient effect on proliferation and maturation of erythropoietic cells in the rat bone marrow and the sporadic appearance of micronucleated immature bone marrow red cells.     

Hepatoprotective effect of L-carnitine against acute acetaminophen toxicity in mice.

L-carnitine is a cofactor in the transfer of long-chain fatty acid allowing the beta-oxidation of fatty acid in the mitochondria. It is also a known antioxidant with protective effects against lipid peroxidation. In this study, hepatoprotective effect of L-carnitine was investigated against acetaminophen (AA)-induced liver toxicity where mitochondrial dysfunction and oxidative stress are thought to be involved in AA hepatotoxicity. Sixty-four Balb/C mice were divided into eight groups. Mice were dosed with single-AA injection (500 mg/kg via the intra peritoneal route) with or without L-carnitine (500 mg/kg for 5 days starting 5 days before AA injection via intra peritoneal route) and sampled at 4, 8 and 24 h following AA injection. AA increased serum AST, ALT, total sialic acid (TSA) and MDA as well as tissue TSA and MDA levels significantly with the highest increase observed at 4 h, but there was a decrease in blood and tissue GSH level. Administration of L-carnitine significantly reduced AA-induced elevations in AST, ALT, TSA and MDA concentrations and increased GSH levels at all sampling points. AA also induced necrosis, hyperemia, sinusoidal congestion and hemorrhage with time-dependent increase in severity, but the degree of necrosis and histopathologic alterations were most severe at 24 h following AA administration. However, the degree of pathologic alterations was less severe with simultaneous L-carnitine application. These results suggest that AA results in oxidative damage in the liver with an acute effect. L-carnitine also has a prominent protective effect against AA toxicity and may be of therapeutic value in the treatment of AA-induced hepatotoxicity.     

Resting and pure tone evoked metabolic responses in the inferior colliculus of young adult and senescent rats

Metabolic responses from three sectors of the inferior colliculus (IC) divided according to frequency representation were examined bilaterally in young (YA) and aged adult (AA) rats using the 2-deoxyglucose method under quiescent or pure tone stimulus conditions. Resting IC metabolism in YA rats is characterized by elevated glucose uptake in the ventromedial (high frequency) relative to intermediate and dorsolateral sectors. AA resting incorporation is not reduced in any sector and is more uniform than YA uptake. Monaural high frequency stimulation (50 kHz) in YA animals evokes discrete contralateral banding along the ventromedial IC border, while stimulation in the optimal auditory sensitivity range of the rat at 8 kHz activates the intermediate sector of the contralateral IC. Although 50 kHz evoked ventromedial uptake is slightly reduced in AA rats contralaterally, the overall uptake within the three sectors does not differ significantly between ages under either of the monaural stimulus conditions; therefore senescent stability is exhibited. However, in AA rats the focus to 8 kHz stimulation has less clearly defined borders even though uptake within the intermediate sector is undiminished compared to YA animals, while 50 kHz elicited activity is typically reduced to a discontinuous band in the ventromedial sector of AA animals. These senescent changes may represent compensation in IC metabolism to changes in cochlear processing which are reflected in the activity of ascending auditory pathways.     

Chronic uridine treatment reduces the level of [3H]spiperone-labelled dopamine receptors and enhances their turnover rate in striatum of young rats: relationship to dopamine-dependent behaviours

The effect was studied of chronic uridine treatment on the recovery of striatal D-2 dopamine (DA) receptors after their irreversible blockade by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in young (40 days old) and adult (14 months old) male rats using [³H]spiperone as radioligand. Chronic uridine treatment (15 mg kg^-1 day^-1, i.p., 14 days) causes a reduction of [³H]spiperone binding sites in striatum of young rats. This treatment also produces an increase in the rate of recovery of striatal [³H]spiperone-labelled DA receptors in young, but not in adult rats. Catalepsy and exploratory locomotor activity, two behaviours associated with blockade versus activation of DA receptors, were evaluated in the same rats. The behavioural recovery from the EEDC^induced syndrome is more rapid in the young rats treated with uridine than in the saline-treated group. The behavioural recovery in old rats was not affected by chronic uridine treatment. Thus, in young rats the pyrimidine nucleoside uridine may modulate the steady state and the turnover rate of striatal D-2 DA receptors.     

Nippostrongylus brasiliensis in young rats. Lymphocytes expel larval infections but not adult worms.

Young rats were not able to expel adult N. brasiliensis infections even when the worms were damaged by antibodies and the young rats were given all the cellular components (sensitized lymphocytes and bone marrow cells) shown to be necessary for the expulsion of antibody-damaged worms from adult rats. In contrast, most of the worms were expelled from young rats given sensitized lymph node cells on the day of a larval infection. These results show that the reduced ability of young rats to respond to infection by producing sensitized lymphocytes only partly explains their inability to expel the worms. It was not possible to explain the failure of young rats to expel adult worms by hypothesizing that they develop an active factor which prevents the cells from acting on the worms. It is also unlikely that worms persist in young rats because they differ in their susceptibility to cells compared with antibody-damaged worms from mature rats. This work suggests that the immune mechanism which affects the immature stages of this nematode may differ from that which controls the adult stages.     

Antigenotoxic Effect of Ferulic Acid in 7,12-Dimethyl Benz(a)-Anthracene (DMBA) Induced Genotoxicity

The antigenotoxic effect of ferulic acid was carried out by evaluating the cytogenetic markers, the micronuclei frequency and chromosomal aberrations, in the bone marrow of hamsters in 7,12-dimethylbenz(a)anthracene (DMBA) induced genotoxicity. Genotoxicity was induced in experimental hamsters by single intraperitoneal injection of DMBA (30mg kg⁻¹ b.w). Pretreatment of ferulic acid orally at a dose of 40mg kg⁻¹ b.w for five days significantly reduced the frequency of micronucleated polychromatic erythrocytes (MnPCEs) and the percentage of chromosomal aberrations in hamster''s bone marrow. Our results thus suggest that ferulic acid has potent antigenotoxic effect in DMBA induced genotoxicity in golden Syrian hamsters.     

DNA repair deficiency and acetaldehyde-induced chromosomal alterations in CHO cells

Induction of chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) by acetaldehyde (AA) was evaluated in parental and different DNA repair-deficient Chinese hamster ovary (CHO) cell lines to elucidate the mechanisms involved in the protection against AA-induced chromosome damage. Cell lines employed included the parental (AA8), nucleotide excision repair (UV4, UV5, UV61), base excision repair (EM9), homologous recombination repair (HRR) (irs1SF, 51D1)-deficient and Fanconi-like (KO40) ones. The ranking of different cell lines for sensitivity to induction of CAs by AA was 51D1 > irs1SF > KO40 > UV4 > V33-EM9-AA8 > UV61-UV5 in a descending order. Cells deficient in HRR were most sensitive followed by Fanconi anaemia like (KO40) suggesting these pathways, especially HRR is very important for the repair of AA-induced lesions. These observations also suggest that interstrand cross links are primary biologically relevant DNA lesions induced by AA for induction of CAs. Only marginal differences were found between the cell lines for induction of SCEs. The possible mechanisms involved in AA-induced chromosomal alterations are discussed.     

Age-specific changes in expression, activity, and activation of the c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinases by methyl methanesulfonate in rats

The stress-activated protein kinases (SAPKs), c-Jun NH(2)-terminal kinases (JNKs) and p38 mitogen-activated protein kinases, were evaluated in the liver and brain of young and old rats in response to a direct-acting alkylating agent, methyl methanesulfonate (MMS). A slight but statistically significant increase in the baseline expression levels of JNK isoforms was detected in both the liver and brain of old as compared with young rats. In the liver of both young and old rats, no basal activities of JNKs were detected. In the brain, JNK activities were constitutively high and significantly increased in old rats compared with their young counterparts. Upon MMS treatment, JNKs were strongly activated in the liver, but not in the brain, of both young and old animals. The basal activity of p38 significantly increased in both the liver and brain of old rats as compared with young rats. An increase in the basal expression of p38 was detected in the brain but not in the liver of old rats. Upon treatment with MMS, p38 was activated in the liver of both young and old rats. In the brain, p38 was only activated in young but not in old rats. Taken together, these results demonstrate age-specific as well as organ-specific SAPKs signaling pathways in the rat in vivo. The possible implications of these findings in terms of resistance to endogenous and environmentally induced genotoxic stress during aging are discussed.     

Impaired NADPH oxidase activity in Rac2-deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid

Rac2 is a hematopoietic-specific Rho-GTPase that plays a stimulus-specific role in regulating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and other functional responses in neutrophils. In this study, rac2-/- neutrophils were shown to have significantly decreased NADPH oxidase activity and actin remodeling in response to exogenous arachidonic acid (AA), as previously observed for phorbol 12-myristate 13-acetate (PMA) or formyl-Met-Leu-Phe (fMLP) as agonists. PMA-, fMLP-, or AA-induced translocation of p47phox and p67phox to the plasma membrane was not impaired in rac2-/- neutrophils. Combined stimulation of rac2-/- neutrophils with exogenous AA and PMA had a synergistic effect on NADPH oxidase activity, and superoxide production increased to a level that was at least as high as wild-type cells and had no effect on fMLP-elicited enzyme activity. Membrane translocation of p47phox and p67phox as well as Rac1 activation was not increased further by combined PMA and AA stimulation. Inhibitor studies were consistent with important roles for phorbol ester-activated protein kinase C (PKC) isoforms and an atypical isoform, PKCzeta, in superoxide production by wild-type and rac2-/- neutrophils stimulated with AA and PMA. In addition, PMA-stimulated release of AA and cytoplasmic phospholipase A2 expression in rac2-/- neutrophils were similar to wild-type, suggesting that deficient AA production by PMA-stimulated rac2-/- neutrophils does not explain the effect of exogenous AA on oxidase activity. Although not required for translocation of p47phox and p67phox, Rac2 is necessary for optimal activity of the assembled oxidase complex, an effect that can be replaced by exogenous AA, which may act directly or via an exogenous AA-induced mediator.