Systemic absorption of benzalkonium chloride after maximal use of a consumer antiseptic wash product.

An in vivo pharmacokinetic study was conducted using consumer antiseptic wash containing 0.13% benzalkonium chloride (BAC) to assess the effect of dermal absorption on long-term systemic exposure to BAC. The objective of the study was to determine blood levels of BAC under maximal use conditions. Subjects were enlisted to wash their hands 60 s with soap containing 0.13% BAC 30 times per day over an 8-9 h time period for 5 consecutive days. The test product with the highest absorption potential was selected based on market share and results from in vitro permeation testing. Blood plasma was collected from subjects on 32 occasions over the 6-day study period. Plasma samples were analyzed for the C12 and C14 homologs of BAC using LC-MS/MS with a lower limit of quantitation (LLOQ) of 106.9 and 32.6 ng/L, respectively. For the 32 subjects, C12 homolog was detected above the LLOQ in only four of 1,024 plasma samples at 117.8-191.7 ng/L, and C14 homolog was detected in only one sample at 59.5 ng/L. Consequently, systemic exposure to BAC in antimicrobial soap is very low and below the level of concern identified by the U.S. Food and Drug Administration (500 ng/L) even under maximal use conditions.

Involvement of oxidants and AP-1 in angiotensin II-activated NFAT3 transcription factor.

Cardiomyocyte hypertrophy is associated with multiple pathophysiological cardiovascular conditions. Recent studies have substantiated the finding that oxidants may contribute to the development of cardiomyocyte hypertrophy. Activation of the nuclear factor of activated T cells-3 (NFAT3) transcription factor has been shown to result from endocrine inducers of cardiomyocyte hypertrophy such as angiotensin II (ANG II) and serves as an important molecular regulator of cardiomyocyte hypertrophy. In this study, we found that antioxidant enzyme catalase and antioxidants N-acetyl-l-cysteine, alpha-phenyl-N-tert-butylnitrone, and lipoic acid prevent ANG II from activating NFAT3 promoter-luciferase. H(2)O(2) induces a time- and dose-dependent activation of NFAT3 transcription factor. A dominant negative form of NFAT3 transcription factor inhibited H(2)O(2) from activating NFAT3 promoter. An inhibitor of ERKs, but not phosphoinositide 3-kinase or p38 MAPKs, blocked NFAT3 activation by H(2)O(2). The NFAT3 binding site in the promoters of most genes contains a weak activator protein-1 (AP-1) binding site adjacent to the core consensus NFAT binding sequence. ERK inhibitor PD98059 was found previously to inhibit AP-1 activation by H(2)O(2). Inactivation of AP-1 transcription factor by cotransfection of a dominant negative c-Jun, TAM67, prevented H(2)O(2) or ANG II from activating NFAT3 promoter. NFAT3 promoter containing the core NFAT cis-element without AP-1 binding site failed to show activation by H(2)O(2) treatment. Our data suggest that hypertrophy inducers ANG II and H(2)O(2) may activate NFAT3 in cardiomyocyte through an AP-1 transcription factor-dependent mechanism.

Distinct roles of p42/p44(ERK) and p38 MAPK in oxidant-induced AP-1 activation and cardiomyocyte hypertrophy.

Cardiac hypertrophy is an adaptive response to a number of heart diseases including myocardial infarction. Although it can be compensatory at first, sustained hypertrophy is often a transition to heart failure. We have found that cardiomyocytes in culture can survive mild doses of H2O2 but develop hypertrophy involving activation of p70 S6 kinase 1 (p70S6K1). Here, the role of p42/p44(ERK) and p38 MAPK in oxidant-induced hypertrophy is tested. H2O2- induced phosphorylation (activation) of p42/p44(ERK) and p38 within 10 min of 200 microM H2O2 exposure. Although p42/p44(ERK) remained highly phosphorylated from 60 to 120 min, the level of p38 phosphorylation reached highest at 60 min and started to decline at 90 min. Inhibiting ERKs with PD98059 attenuated H2O2-induced AP-1 activation but did not affect H2O2-induced p70S6K1 activation or cardiomyocyte enlargement as measured by increases in cell volume and protein content. In contrast, the p38 inhibitor SB202190 has no inhibitory effect on AP-1 activation but partially prevented H2O2 from inducing p70S6K1 activation and cell enlargement. These data suggest that while p42/p44(ERK) participates in gene expression associated with hypertrophy, p38 may regulate cell size increase by p70S6K1 activation.

Signals of oxidant-induced cardiomyocyte hypertrophy: key activation of p70 S6 kinase-1 and phosphoinositide 3-kinase.

Cardiomyocytes in culture can survive low or mild doses of oxidants but later increase cell volume and protein content. To understand the mechanism, we determined the early signaling events of oxidative stress. With 200 microM H2O2, the activity of p70 S6 kinase-1 (p70S6K1) increased at 30 min and reached a plateau at 90 min. Dose-response studies at the 60 min time point show that p70S6K1 activity reached its highest level with 150 microM H2O2. Increased p70S6K1 activity correlated with phosphorylation of Thr389 and Thr421/Ser424 residues, suggesting the involvement of an upstream kinase. Phosphoinositide 3-kinase (PI3K) activity was elevated by 5 min, reached a plateau at 10 min, and remained more than 6-fold induced for at least 60 min after 200 microM H2O2 exposure. The dose-response studies at 10 min found that 150 microM H2O2 induced the highest PI3K activity. Increased PI3K activity correlated with tyrosine phosphorylation of the 85-kDa regulatory subunit. Inactivating PI3K with wortmannin prevented H2O2 from inducing Thr389 phosphorylation and p70S6K1 activation. Wortmannin and rapamycin prevented H2O2 from inducing increases in cell volume and protein content. The antineoplastic drugs doxorubicin and daunorubicin also induced significant enlargement of cardiomyocytes at 10 to 100 nM dose range. Although the glutathione synthesis inhibitor buthionine sulfoximine potentiated the effect of doxorubicin and H2O2, the antioxidant N-acetylcysteine prevented induction of cell enlargement. Our data suggest that oxidative stress induces activation of PI3K, which leads to p70S6K1 activation and enlargement of cell size.

Apoptosis and heart failure: mechanisms and therapeutic implications.

A large volume of experimental data supports the presence of apoptosis in failing hearts. Apoptosis in many types of cells results from exposure to cytotoxic cytokines or damaging agents. Cytotoxic cytokines such as tumor necrosis factor (TNF)-alpha or Fas ligand (FasL) bind to their receptors to activate caspase-8, while damaging agents can cause mitochondrial release of cytochrome c, which can initiate activation of caspase-9. Caspase-8 or -9 can activate a cascade of caspases. The p53 protein is often required for damaging agent-induced apoptosis. An imbalance of proapoptotic factors versus prosurvival factors in the bcl-2 family precedes the activation of caspases. Given these typical changes of apoptosis found in many cell types, the apoptotic pathway in cardiomyocytes is somewhat unconventional since in vivo experimental data reveal that apoptosis does not appear to be controlled by TNF-alpha, FasL, p53 or decrease of bcl-2. In vitro and in vivo studies suggest the importance of mitochondria and activation of caspases in cell death occurring in failing hearts. Oxidants, excessive nitric oxide, angiotensin II and catecholamines have been shown to trigger apoptotic death of cardiomyocytes. Eliminating these inducers reduces apoptosis and reverses the loss of contractile function in many cases, indicating the feasibility of the pharmacological application of antioxidants, nitric oxide synthetase inhibitors, ACE inhibitors, angiotensin II receptor antagonists and adrenergic receptor antagonists. Most inducers of apoptosis initiate a cascade of signaling events, including activation of the p38 mitogen-activated protein kinase. Small molecule inhibitors of p38 have been shown to be capable of preventing apoptosis and loss of contractile function associated with ischemia and reperfusion. Although further experimental work is needed, several studies have already indicated the beneficial effect of caspase inhibitors against cell loss and features of heart failure in vitro and in vivo. These studies indicate the importance of inhibiting apoptosis in therapeutic interventions against heart failure.