Chronic, topical exposure to benzo[a]pyrene induces relatively high steady-state levels of DNA adducts in target tissues and alters kinetics of adduct loss.

Carcinogen-DNA adduct measurements may become useful biomarkers of effective dose and/or early effect. However, validation of this biomarker is required at several levels to ensure that human exposure and response are accurately reflected. Important in this regard is an understanding of the relative biomarker levels in target and nontarget organs and the response of the biomarker under the chronic, low-dose conditions to which humans are exposed. We studied the differences between single and chronic topical application of benzo[a]pyrene (BAP) on the accumulation and removal of BAP-DNA adducts in skin, lung, and liver. Animals were treated with BAP at 10, 25, or 50 nMol topically once or twice per week for as long as 15 weeks. Animals were sacrificed either at 24, 48, or 72 hr after the last dose at 1 and 30 treatments, and after 24 hr for all other treatment groups. Adduct levels increased with increasing dose, but the slope of the dose-response was different in each organ. At low doses, accumulation was linear in skin and lung, but at high doses the adduct levels in the lung increased dramatically at the same time when the levels in the skin reached apparent steady state. In the liver adduct, levels were lower than in target tissues and apparent steady-state adduct levels were reached rapidly, the maxima being independent of dose, suggesting that activating metabolism was saturated in this organ. Removal of adducts from skin, the target organ, was more rapid following single treatment than with chronic exposure. This finding is consistent with earlier data, indicating that some areas of the genome are more resistant to repair. Thus, repeated exposure and repair cycles would be more likely to cause an increase in the proportion of carcinogen-DNA adducts in repair-resistant areas of the genome. These findings indicate that single-dose experiments may underestimate the potential for carcinogenicity for compounds that follow this pattern.

Inactivation of tyrosine hydroxylase by nitration following exposure to peroxynitrite and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

The decrement in dopamine levels exceeds the loss of dopaminergic neurons in Parkinson’s disease (PD) patients and experimental models of PD. This discrepancy is poorly understood and may represent an important event in the pathogenesis of PD. Herein, we report that the rate-limiting enzyme in dopamine synthesis, tyrosine hydroxylase (TH), is a selective target for nitration following exposure of PC12 cells to either peroxynitrite or 1-methyl-4-phenylpyridiniun ion (MPP+). Nitration of TH also occurs in mouse striatum after MPTP administration. Nitration of tyrosine residues in TH results in loss of enzymatic activity. In the mouse striatum, tyrosine nitration-mediated loss in TH activity parallels the decline in dopamine levels whereas the levels of TH protein remain unchanged for the first 6 hr post MPTP injection. Striatal TH was not nitrated in mice overexpressing copper/zinc superoxide dismutase after MPTP administration, supporting a critical role for superoxide in TH tyrosine nitration. These results indicate that tyrosine nitration-induced TH inactivation and consequently dopamine synthesis failure, represents an early and thus far unidentified biochemical event in MPTP neurotoxic process. The resemblance of the MPTP model with PD suggests that a similar phenomenon may occur in PD, influencing the severity of parkisonian symptoms.

Developmental exposure to vasopressin increases aggression in adult prairie voles

Although the biological roots of aggression have been the source of intense debate, the precise physiological mechanisms responsible for aggression remain poorly understood. In most species, aggression is more common in males than females; thus, gonadal hormones have been a focal point for research in this field. Although gonadal hormones have been shown to influence the expression of aggression, in many cases aggression can continue after castration, indicating that testicular steroids are not completely essential for the expression of aggression. Recently, the mammalian neuropeptide arginine vasopressin (AVP) has been implicated in aggression. AVP plays a particularly important role in social behavior in monogamous mammals, such as prairie voles (Microtus ochrogaster). In turn, the effects of social experiences may be mediated by neuropeptides, including AVP. For example, sexually naïve prairie voles are rarely aggressive. However, 24 h after the onset of mating, males of this species become significantly aggressive toward strangers. Likewise, in adult male prairie voles, central (intracerebroventricular) injections of AVP can significantly increase intermale aggression, suggesting a role for AVP in the expression of postcopulatory aggression in adult male prairie voles. In this paper, we demonstrate that early postnatal exposure to AVP can have long-lasting effects on the tendency to show aggression, producing levels of aggression in sexually naïve, adult male prairie voles that are comparable to those levels observed after mating. Females showed less aggression and were less responsive to exogenous AVP, but the capacity of an AVP V1a receptor antagonist to block female aggression also implicates AVP in the development of female aggression.

Cardioprotection from ischemia by a brief exposure to physiological levels of ethanol: Role of epsilon protein kinase C

Recent epidemiological studies indicate beneficial effects of moderate ethanol consumption in ischemic heart disease. Most studies, however, focus on the effect of long-term consumption of ethanol. In this study, we determined whether brief exposure to ethanol immediately before ischemia also produces cardioprotection. In addition, because protein kinase C (PKC) has been shown to mediate protection of the heart from ischemia, we determined the role of specific PKC isozymes in ethanol-induced protection. We demonstrated that (i) brief exposure of isolated adult rat cardiac myocytes to 10–50 mM ethanol protected against damage induced by prolonged ischemia; (ii) an isozyme-selective ɛPKC inhibitor developed in our laboratory inhibited the cardioprotective effect of acute ethanol exposure; (iii) protection of isolated intact adult rat heart also occurred after incubation with 10 mM ethanol 20 min before global ischemia; and (iv) ethanol-induced cardioprotection depended on PKC activation because it was blocked by chelerythrine and GF109203X, two PKC inhibitors. Consumption of 1–2 alcoholic beverages in humans leads to blood alcohol levels of ≈10 mM. Therefore, our work demonstrates that exposure to physiologically attainable ethanol levels minutes before ischemia provides cardioprotection that is mediated by direct activation of ɛPKC in the cardiac myocytes. The potential clinical implications of our findings are discussed.

Gestational exposure to ethanol suppresses msx2 expression in developing mouse embryos

Ethanol acts as a teratogen in developing fetuses causing abnormalities of the brain, heart, craniofacial bones, and limb skeletal elements. To assess whether some teratogenic actions of ethanol might occur via dysregulation of msx2 expression, we examined msx2 expression in developing mouse embryos exposed to ethanol on embryonic day (E) 8 of gestation and subjected to whole mount in situ hybridization on E11–11.5 using a riboprobe for mouse msx2. Control mice exhibited expression of msx2 in developing brain, the developing limb buds and apical ectodermal ridge, the lateral and nasal processes, olfactory pit, palatal shelf of the maxilla, the eye, the lens of the eye, otic vesicle, prevertebral bodies (notochord), and endocardial cushion. Embryos exposed to ethanol in utero were significantly smaller than their normal counterparts and did not exhibit expression of msx2 in any structures. Similarly, msx2 expression, as determined by reverse transcription–PCR and Northern blot hybridization, was reduced ≈40–50% in fetal mouse calvarial osteoblastic cells exposed to 1% ethanol for 48 hr while alkaline phosphatase was increased by 2-fold and bone morphogenetic protein showed essentially no change. Transcriptional activity of the msx2 promoter was specifically suppressed by alcohol in MC3T3-E1 osteoblasts. Taken together, these data demonstrate that fetal alcohol exposure decreases msx2 expression, a known regulator of osteoblast and myoblast differentiation, and suggest that one of the “putative” mechanisms for fetal alcohol syndrome is the inhibition of msx2 expression during key developmental periods leading to developmental retardation, altered craniofacial morphogenesis, and cardiac defects.

Long-term exposure to high corticosterone levels attenuates serotonin responses in rat hippocampal CA1 neurons

Recent studies indicated that hyperactivity of the hypothalamo-pituitary-adrenal system is a considerable risk factor for the precipitation of affective disorders, most notably of major depression. The mechanism by which this hyperactivity eventually leads to clinical symptoms of depression is unknown. In the present animal study, we tested one possible mechanism, i.e., that long-term exposure to high corticosterone levels alters functional responses to serotonin in the hippocampus, an important area in the etiology of depression. Rats were injected daily for 3 weeks with a high dose of corticosterone; electrophysiological responses to serotonin were recorded intracellularly from CA1 pyramidal neurons in vitro. We observed that daily injections with corticosterone gradually attenuate the membrane hyperpolarization and resistance decrease mediated by serotonin-1A receptors. We next used single-cell antisense RNA amplification from identified CA1 pyramidal neurons to resolve whether the functional deficits in serotonin responsiveness are accompanied by decreased expression levels of the serotonin-1A receptor. It appeared that expression of serotonin-1A receptors in CA1 pyramidal cells is not altered; this result was supported by in situ hybridization. Expression of corticosteroid receptors in the same cells, particularly of the high-affinity mineralocorticoid receptor, was significantly reduced after long-term corticosterone treatment. The present findings indicate that prolonged elevation of the corticosteroid concentration, a possible causal factor for major depression in humans, gradually attenuates responsiveness to serotonin without necessarily decreasing serotonin-1A receptor mRNA levels in pyramidal neurons. These functional changes may occur by a posttranscriptional mechanism or by transcriptional regulation of genes other than the serotonin-1A receptor gene itself.

Anti-CD14 mAb treatment provides therapeutic benefit after in vivo exposure to endotoxin

The presence of endotoxin from Gram-negative bacteria signals the innate immune system to up-regulate bacterial clearance and/or killing mechanisms. Paradoxically, such responses also contribute to septic shock, a clinical problem occurring with high frequency in Gram-negative septicemia. CD14 is a receptor for endotoxin (lipopolysaccharide, LPS) and is thought to have an essential role in innate immune responses to infection and thereby in the development of septic shock. Using a novel rabbit model of endotoxic shock produced by multiple exposures to endotoxin, we show that anti-rabbit CD14 mAb, which blocks LPS-CD14 binding, protects against organ injury and death even when the antibody is administered after initial exposures to LPS. In contrast, anti-rabbit tumor necrosis factor mAb treatment fails to protect when administered after LPS injections. These results support the concept that anti-CD14 treatment provides a new therapeutic window for the prevention of pathophysiologic changes that result from cumulative exposures to LPS during septic shock in man.

Early-life exposure to endotoxin alters hypothalamic–pituitary–adrenal function and predisposition to inflammation

We have investigated whether exposure to Gram-negative bacterial endotoxin in early neonatal life can alter neuroendocrine and immune regulation in adult animals. Exposure of neonatal rats to a low dose of endotoxin resulted in long-term changes in hypothalamic–pituitary–adrenal (HPA) axis activity, with elevated mean plasma corticosterone concentrations that resulted from increased corticosterone pulse frequency and pulse amplitude. In addition to this marked effect on the development of the HPA axis, neonatal endotoxin exposure had long-lasting effects on immune regulation, including increased sensitivity of lymphocytes to stress-induced suppression of proliferation and a remarkable protection from adjuvant-induced arthritis. These findings demonstrate a potent and long-term effect of neonatal exposure to inflammatory stimuli that can program major changes in the development of both neuroendocrine and immunological regulatory mechanisms.

Autoinhibition of serotonin cells: An intrinsic regulatory mechanism sensitive to the pattern of usage of the cells

After periods of high-frequency firing, the normal rhythmically active serotonin (5HT)-containing neurosecretory neurons of the lobster ventral nerve cord display a period of suppressed spike generation and reduced synaptic input that we refer to as “autoinhibition.” The duration of this autoinhibition is directly related to the magnitude and duration of the current injection triggering the high-frequency firing. More interesting, however, is that the autoinhibition is inversely related to the initial firing frequency of these cells within their normal range of firing (0.5–3 Hz). This allows more active 5HT neurons to resume firing after shorter durations of inhibition than cells that initially fired at slower rates. Although superfused 5HT inhibits the spontaneous firing of these cells, the persistence of autoinhibition in saline with no added calcium, in cadmium-containing saline, and in lobsters depleted of serotonin suggests that intrinsic membrane properties account for the autoinhibition. A similar autoinhibition is seen in spontaneously active octopamine neurons but is absent from spontaneously active γ-aminobutyric acid cells. Thus, this might be a characteristic feature of amine-containing neurosecretory neurons. The 5HT cells of vertebrate brain nuclei share similarities in firing frequencies, spike shapes, and inhibition by 5HT with the lobster cells that were the focus of this study. However, the mechanism suggested to underlie autoinhibition in vertebrate neurons is that 5HT released from activated or neighboring cells acts back on inhibitory autoreceptors that are found on the dendrites and cell bodies of these neurons.