Chloride extrusion enhancers as novel therapeutics for neurological diseases.

The K(+)-Cl(-) cotransporter KCC2 is responsible for maintaining low Cl(-) concentration in neurons of the central nervous system (CNS), which is essential for postsynaptic inhibition through GABA(A) and glycine receptors. Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain. Recent reports indicate that enhancing KCC2 activity may be the favored therapeutic strategy to restore inhibition and normal function in pathological conditions involving impaired Cl(-) transport. We designed an assay for high-throughput screening that led to the identification of KCC2 activators that reduce intracellular chloride concentration ([Cl(-)]i). Optimization of a first-in-class arylmethylidine family of compounds resulted in a KCC2-selective analog (CLP257) that lowers [Cl(-)]i. CLP257 restored impaired Cl(-) transport in neurons with diminished KCC2 activity. The compound rescued KCC2 plasma membrane expression, renormalized stimulus-evoked responses in spinal nociceptive pathways sensitized after nerve injury and alleviated hypersensitivity in a rat model of neuropathic pain. Oral efficacy for analgesia equivalent to that of pregabalin but without motor impairment was achievable with a CLP257 prodrug. These results validate KCC2 as a druggable target for CNS diseases.

Quantitative approaches for assessing dose-response relationships in genetic toxicology studies.

Genetic toxicology studies are required for the safety assessment of chemicals. Data from these studies have historically been interpreted in a qualitative, dichotomous "yes" or "no" manner without analysis of dose-response relationships. This article is based upon the work of an international multi-sector group that examined how quantitative dose-response relationships for in vitro and in vivo genetic toxicology data might be used to improve human risk assessment. The group examined three quantitative approaches for analyzing dose-response curves and deriving point-of-departure (POD) metrics (i.e., the no-observed-genotoxic-effect-level (NOGEL), the threshold effect level (Td), and the benchmark dose (BMD)), using data for the induction of micronuclei and gene mutations by methyl methanesulfonate or ethyl methanesulfonate in vitro and in vivo. These results suggest that the POD descriptors obtained using the different approaches are within the same order of magnitude, with more variability observed for the in vivo assays. The different approaches were found to be complementary as each has advantages and limitations. The results further indicate that the lower confidence limit of a benchmark response rate of 10% (BMDL(10) ) could be considered a satisfactory POD when analyzing genotoxicity data using the BMD approach. The models described permit the identification of POD values that could be combined with mode of action analysis to determine whether exposure(s) below a particular level constitutes a significant human risk. Subsequent analyses will expand the number of substances and endpoints investigated, and continue to evaluate the utility of quantitative approaches for analysis of genetic toxicity dose-response data.

Hepatocellular proliferation and hepatocarcinogen bioactivation in mice with diet-induced fatty liver and obesity.

Human liver cancer is in part associated with obesity and related metabolic diseases. The present study was undertaken in a mouse model of diet-induced obesity (DIO) and hepatic steatosis, conditions which can be associated with hepatic neoplasia, to determine whether the rates of cell proliferation or hepatocarcinogen bioactivation were altered in ways which could facilitate hepatocarcinogenesis. DIO mice were generated by feeding C57BL/6 (B6) male mice a high-fat diet beginning at 4 weeks of age; age-matched conventional lean (LEAN) B6 mice fed a low fat diet (10% Kcal from fat) were used for comparison. Groups of 28 week old DIO and LEAN mice were dosed with the bioactivation-dependent DNA-reactive hepatocarcinogen 2-acetylaminofluorene (AAF), at 2.24 or 22.4 mg/kg, given by gavage 3 times per week for 31 days, or received no treatment (DIO and LEAN control groups). Compared with the LEAN control group, the DIO control group had a higher mean body weight (16.5 g), higher mean absolute (1.4 g) and mean relative (25.5%) liver weights, higher (394%) liver triglyceride concentrations, and an increased incidence and severity of hepatocellular steatosis at the end of the dosing phase. The DIO control group also had a higher mean hepatocellular replicating fraction (31% increase, determined by proliferating cell nuclear antigen immunohistochemistry). Hepatocarcinogen bioactivation, based on formation of AAF DNA adducts as measured by nucleotide (32)P-postlabeling, was similar in both DIO and LEAN AAF-dosed groups. Thus, hepatocellular proliferation, but not hepatocarcinogen bioactivation, was identified as an alteration in livers of DIO mice which could contribute to their susceptibility to hepatocarcinogenesis.

The manipulation of cation-chloride co-transporters as a novel means to treat persistent pain, epilepsy and other neurological disorders.

Existing members of the anti-epileptic drug (AED) class often fail to provide meaningful symptom relief to patients experiencing persistent pain, epilepsy and other neurological disorders, and can evoke substantial adverse events. In an effort to improve treatment options, much attention has turned to novel mechanisms that may represent points of therapeutic intervention. Among these mechanisms are the cation-chloride co-transporters (CCCs), the dysfunction of which has been linked to aberrant chloride homeostasis in neurons of the CNS, and resulting disorders including persistent pain and epilepsy. This review examines the literature linking CCCs to neurological disease, and discusses their considerable potential as the basis for novel therapeutic strategies.

Multicenter study to assess potential hazards from exposure to lipid peroxidation products in soya bean oil from Trilucent breast implants.

In response to a Hazard Notice by the Medical Devices Agency of the UK in 2000 regarding the Trilucent breast implant (TBI), an expert panel was convened to implement a research program to determine whether genotoxic compounds were formed in the soybean oil filler (SOF) of TBIs and whether these could be released to produce local or systemic genotoxicity. The panel established a research program involving six laboratories. The program recruited 47 patients who had received TBIs (9 patients had received silicone implants previously). A reference group (REBI) of 34 patients who had exchanged either silicone (17 patients) implants (REBI-E) or patients (17) who were to receive primary implantation augmentation with silicone (REBI-PIA), and who were included as needed to increase either the pre- or post-explantation sample number. Of the 17 REBI-E patients, 5 had silicone implants and 12 had saline implants previously (prior to the last exchange). Investigation was undertaken before and after replacement surgery in the TBI patients and before and after replacement or augmentation surgery in the REBI patients. The pre- to post-operative sample interval was 8-12 weeks. Pre-operative samples were collected within 7 days prior to the operation. Information on a variety of demographic and behavioral features was collected. Biochemical and biological endpoints relating to genotoxic lipid peroxidation (LPO) products potentially formed in the SOF, and released locally or distributed systemically, were measured. The SOF of explanted TBIs was found to have substantial levels of LPO products, particularly malondialdehyde (MDA), and low levels of trans-4-hydroxy-2-nonenal (HNE) not found in unused implants. Mutagenicity of the SOF was related to the levels of MDA. Capsules that formed around TBIs were microscopically similar to those of reference implants, but MDA-DNA adducts were observed in capsular macrophages and fibroblasts of only TBI capsules. These cell types are not progenitors of breast carcinoma (BCa) and the location of the implants precludes LPO products reaching the mammary epithelial cells which are progenitors of BCa. Blood levels of LPO products were not increased in TBI patients compared to REBI patients and did not change with explantation. In TBI patients, white blood cells did not show evidence of increased levels of LPO-related aldehyde DNA adducts. In conclusion, based on a number of measured parameters, there was no evident effect that would contribute to breast or systemic cancer risk in the TBI patients, and the recommended treatment of TBI patients involving explantation was judged appropriate.

BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain.

Neuropathic pain that occurs after peripheral nerve injury depends on the hyperexcitability of neurons in the dorsal horn of the spinal cord. Spinal microglia stimulated by ATP contribute to tactile allodynia, a highly debilitating symptom of pain induced by nerve injury. Signalling between microglia and neurons is therefore an essential link in neuropathic pain transmission, but how this signalling occurs is unknown. Here we show that ATP-stimulated microglia cause a depolarizing shift in the anion reversal potential (E(anion)) in spinal lamina I neurons. This shift inverts the polarity of currents activated by GABA (gamma-amino butyric acid), as has been shown to occur after peripheral nerve injury. Applying brain-derived neurotrophic factor (BDNF) mimics the alteration in E(anion). Blocking signalling between BDNF and the receptor TrkB reverses the allodynia and the E(anion) shift that follows both nerve injury and administration of ATP-stimulated microglia. ATP stimulation evokes the release of BDNF from microglia. Preventing BDNF release from microglia by pretreating them with interfering RNA directed against BDNF before ATP stimulation also inhibits the effects of these cells on the withdrawal threshold and E(anion). Our results show that ATP-stimulated microglia signal to lamina I neurons, causing a collapse of their transmembrane anion gradient, and that BDNF is a crucial signalling molecule between microglia and neurons. Blocking this microglia-neuron signalling pathway may represent a therapeutic strategy for treating neuropathic pain.

Differential maturation of GABA action and anion reversal potential in spinal lamina I neurons: impact of chloride extrusion capacity.

A deficit in inhibition in the spinal dorsal horn has been proposed to be an underlying cause of the exaggerated cutaneous sensory reflexes observed in newborn rats. However, the developmental shift in transmembrane anion gradient, potentially affecting the outcome of GABAA transmission, was shown to be completed within 1 week after birth in the spinal cord, an apparent disparity with the observation that reflex hypersensitivity persists throughout the first 2-3 postnatal weeks. To further investigate this issue, we used several approaches to assess the action of GABA throughout development in spinal lamina I (LI) neurons. GABA induced an entry of extracellular calcium in LI neurons from postnatal day 0 (P0) to P21 rats, which involved T- and N-type voltage-gated calcium channels. Gramicidin perforated-patch recordings revealed that the shift in anion gradient was completed by P7 in LI neurons. However, high chloride pipette recordings demonstrated that these neurons had not reached their adult chloride extrusion capacity by P10-P11. Simultaneous patch-clamp recordings and calcium imaging revealed that biphasic responses to GABA, consisting of a primary hyperpolarization followed by a rebound depolarization, produced a rise in [Ca2+]i. Thus, even if Eanion predicts GABAA-induced hyperpolarization from rest, a low chloride extrusion capacity can cause a rebound depolarization and an ensuing rise in [Ca2+]i. We demonstrate that GABA action in LI neurons matures throughout the first 3 postnatal weeks, therefore matching the time course of maturation of withdrawal reflexes. Immature spinal GABA signaling may thus contribute to the nociceptive hypersensitivity in infant rats.

Risk assessment of DNA-reactive carcinogens in food.

Risk assessment of DNA-reactive carcinogens in food requires knowledge of the extent of DNA damage in the target organ which results from the competition between DNA adduct formation and repair. Estimates of DNA adduct levels can be made by direct measurement or indirectly as a consequence of their presence, for example, by tumor formation in animal models or exposed populations epidemiologically. Food-borne DNA-reactive carcinogens are present from a variety of sources. They are generally not intrinsically DNA-reactive but require bioactivation to DNA-reactive metabolites a process which may be modulated by the compound itself or the presence of other xenobiotics. A single DNA reactant may form several distinct DNA adducts each undergoing different rates of repair. Some DNA reactants may be photochemically activated or produce reactive oxygen species and thus indirect oxidative DNA damage. The levels of DNA adducts arising from exposures influenced by variations in the doses, the frequency with which an individual is exposed, and rates of DNA repair for specific adducts. Each adduct has a characteristic efficiency with which it induces mutations. Based on experience with the well-studied DNA-reactive food carcinogen aflatoxin B(1) (AFB(1)), a limit of 20 ppb or approximately 30 microg/day has been set and is considered a tolerable daily intake (TDI). Since AFB(1) is considered a potent carcinogen, doses of <1.5 microg of unknown compounds are considered TDIs. Most DNA-reactants, including acrylamide, heterocyclic amines, and alpha,beta-unsaturated carbonyl are below this value. Above that value, measurement of actual DNA adducts levels in either experimental animals with a risk assessment, or, when this occurs, exposed humans are needed. A number of approaches to undertake this are described including immunological, mass spectrometric and (32)P-postlabeling or the use of surrogates such as hemoglobin adducts, together with approaches to evaluate the results. A discussion of approaches to estimating possible threshold effects for DNA-reactive carcinogens is made.

Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain.

Modern pain-control theory predicts that a loss of inhibition (disinhibition) in the dorsal horn of the spinal cord is a crucial substrate for chronic pain syndromes. However, the nature of the mechanisms that underlie such disinhibition has remained controversial. Here we present evidence for a novel mechanism of disinhibition following peripheral nerve injury. It involves a trans-synaptic reduction in the expression of the potassium-chloride exporter KCC2, and the consequent disruption of anion homeostasis in neurons of lamina I of the superficial dorsal horn, one of the main spinal nociceptive output pathways. In our experiments, the resulting shift in the transmembrane anion gradient caused normally inhibitory anionic synaptic currents to be excitatory, substantially driving up the net excitability of lamina I neurons. Local blockade or knock-down of the spinal KCC2 exporter in intact rats markedly reduced the nociceptive threshold, confirming that the reported disruption of anion homeostasis in lamina I neurons was sufficient to cause neuropathic pain.

Protective effect of acetaminophen against colon cancer initiation effects of 3,2'-dimethyl-4-aminobiphenyl in rats.

A previous investigation demonstrated the anticarcinogenicity of acetaminophen (APAP) against colon carcinogenesis in rats induced by 3,2'-dimethyl-4-aminobiphenyl (DMAB). DMAB was selected as a structurally related surrogate for heterocyclic amines, formed during cooking of protein, which are believed to be involved in human colon cancer. The objective of the present study was to ascertain whether the early initiating effects of this colon carcinogen are inhibited by APAP. Six groups of male F344 rats were treated over a 6-week period as follows: (1) vehicle (corn oil) for 6 weeks; (2) APAP in the diet at 1000 ppm daily for 6 weeks; (3) 50 mg/kg DMAB by gavage once a week for the last 4 weeks; (4) 5 mg/kg DMAB as for (3); (5) 1000 ppm APAP for 6 weeks and 50 mg/kg DMAB for the last 4 weeks; and (6) 1000 ppm APAP and 5 mg/kg DMAB as for (5). Colonic tissue was within normal limits in the control and APAP groups. In the APAP only group, apical enterocytic hypertrophy and hyperaemia over the entire surface epithelium was present. In the high-dose DMAB group, in the lower third of the crypts, foci of enlarged glands with hypertrophic cells exhibiting karyomegaly and anisokaryosis (FHE) of 3+ degree of severity were evident in 100% of the animals. Also, there were increases in periglandular fibrocytes, matrix and mononuclear cells (PF). In the low-dose DMAB group both FHE and PF changes with the same degree of severity were reduced. In rats given the low dose of DMAB plus APAP, FHE and PF with the same degree of severity (3+) was absent. Both DMAB exposures increased significantly the replicating fraction of colonic enterocytes in an exposure-related fashion and the replicating fractions were significantly reduced by APAP. In 32P-postlabelling of colon, liver and urinary bladder DNA, high-dose DMAB produced 2-6 distinct dose-related spots reflecting DNA adducts. These spots were reduced or were no longer detectable in all three tissues when APAP was given 2 weeks before and during DMAB exposure. Using immunohistochemical detection of DMAB adducts in the colon, a dose-related colour intensity was present for both doses of DMAB. APAP reduced this by 94-fold. Thus, APAP produced a marked protective effect in colonic enterocytes against several parameters of neoplastic development by the carcinogen.

Anticarcinogenicity of monocyclic phenolic compounds.

The synthetic monocyclic phenolics (MPs), acetaminophen (APAP), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) are antimutagenic or anticarcinogenic against a diversity of chemical carcinogens affecting a variety of tissues in experimental animals. In studies in this laboratory of the anticarcinogenicity of MPs, the focus has been on delineating efficacy at low levels of MPs that do not elicit adaptive or toxic responses. To accomplish this, we are studying anticarcinogenicity against the neoplastic initiating activity of lower doses of carcinogens than have previously been studied and which are closer to human environmental exposures. In these studies, we have investigated anticarcinogenicity of BHT against liver cancer in rats induced by either 2-acetylaminofluorene (AAF) or aflatoxin B1 (AFB1) and anticarcinogenicity of APAP against colon cancer induced in rats by 3,2'-dimethyl-4-aminobiphenyl (DMAB). BHA and BHT at 100-125 ppm in the diet inhibited the initiation phase of AAF and AFB1 hepatocarcinogenesis and therefore may act intracellularly to block effects of the carcinogen. Likewise, with APAP in colon anticarcinogenicity, at 1000 ppm it reduced DNA binding and exerted a cytoprotective effect against DMAB. Thus, APAP also shows evidence of producing a blocking effect. We conclude that these MPs appear to be anticarcinogenic through a mechanism different from that of most other chemopreventive agents, possibly involving interception of the reactive chemical species of the carcinogen. Accordingly, they have promise as cancer prophylactics, including in combination with agents operating through other mechanisms.

Bioassay of mannitol and caprolactam and assessment of response to diethylnitrosamine in heterozygous p53-deficient (+/-) and wild type (+/+) mice.

Alternative bioassays of mannitol (MAN) and caprolactam (CAP) were conducted in transgenic p53-deficient mice. Also, to assess the sensitivity of the transgenic mice to a model DNA-reactive carcinogen, the hepatic effects of diethylnitrosamine (DEN) were compared in the wild type background strain of mouse and in the transgenic derivative. Fifty-one male wild type strain C57BL/6 mice p53 (+/+), 8 weeks old, and 51 heterozygous p53 (+/-) C57BL/6 Tac-[KO] Trp53 N5 mice, 8 weeks old, were allocated to six experimental groups as follows: groups 1 (wild type +/+) and 2 (p53 +/-) served as room controls, groups 3 (+/+) and 4 (+/-) were exposed orally (gavage) to 50 mumol/kg body weight DEN weekly for a total of ten doses during the first 10 weeks of the study, group 5 (+/-) was exposed to 15,000 ppm CAP in the diet for up to 26 weeks, and group 6 (+/-) was exposed to 50,000 ppm MAN in the diet for up to 26 weeks. After 10 weeks, liver from control and DEN-exposed mice was used for O4-ethylthymidine (O4-EtT) DNA adduct analysis by the immunoslot blot method. The cell replicating fraction (RF) in the liver was determined by quantification of the percentage of immunohistochemically stained hepatocytes positive for proliferating cell nuclear antigen. No significant or consistent body or liver weight changes were present in any of the treatment groups. No consistent and pertinent changes in RF values were present in any of the treatment groups. None of the tested substances produced neoplasms of any type in p53 (+/-) mice. DEN induced comparable levels of O4-EtT adducts in the liver in both wild type and p53 +/- genotypes, but no morphologic changes were evident in the livers of either genotype. The lack of response to DEN, in spite of formation of DNA adducts, may reflect the resistance to hepatocarcinogenesis of the background C57BL/6 strain of the transgenic, and calls into question the general sensitivity of this transgenic for detection of carcinogenic effects.

Assessment of chronic toxicity and carcinogenicity in an accelerated cancer bioassay in rats of moxifloxacin, a quinolone antibiotic.

The chronic toxicity and carcinogenicity of Moxifloxacin (MOX), a bacterial gyrase-inhibiting fluoroquinolone antibiotic, were studied in male and female Wistar rats in an accelerated cancer bioassay (ACB). The ACB is a mechanistic initiation/promotion chronic toxicity and carcinogenicity study designed to assess potential carcinogenic activity of a test substance in critical organs in which human carcinogens are active. The organs studied were liver, lungs, urinary bladder, mammary gland, bone marrow, thymus, spleen and stomach. MOX was given daily by intragastric instillation at 500 mg/kg bw/day for the first 13 weeks to produce potential initiation, followed by promoters (PROs) for 24 weeks, or for the last 24 weeks after 13 weeks of exposure to initiators (INs). The INs, administered during the first 13 weeks, were diethylnitrosamine for the liver, N-n-butyl-N-(4-hydroxybutyl)nitrosamine for the urinary bladder, ethylnitrosourea for the hematolymphoreticular system, N-nitrosodimethylamine for lungs, methylnitrosourea for the stomach and 7,12-dimethylbenz(a)-anthracene for the mammary gland. The PROs, administered during the last 24 weeks after MOX, were phenobarbital for the liver, nitrilotriacetic acid for the urinary bladder, azathioprine for the bone marrow, butylated hydroxytoluene for the lung, butylated hydroxyanisole for the forestomach, and diethylstilbestrol for the mammary gland. The INs produced preneoplastic and neoplastic lesions which were not enhanced by MOX, and MOX plus PROs elicited no neoplastic effects, documenting that MOX did not produce either initiation or promotion of neoplasia in any of the target sites, or in any of the other twenty tissues examined.

Photochemical mutagenicity of phototoxic and photochemically carcinogenic fluoroquinolones in comparison with the photostable moxifloxacin.

Certain fluoroquinolone (FQ) antibiotics that show clinical phototoxicity and experimental photochemical carcinogenicity have been found to interact with ultraviolet-A (UVA) radiation to produce oxidative DNA damage in cultured cells and isolated DNA. To study the biological consequences of oxidative DNA damage in mammalian cells, the photochemical mutagenicity of two photoactive FQs, lomefloxacin and Bay y3118, was studied in V79 cells in comparison with that of the photostable moxifloxacin. Lomefloxacin and Bay y3118 were photochemically mutagenic to V79 cells with UVA irradiation, increasing the mutation frequency by about eightfold (400 microM, 6000 J/m2) and tenfold (50 microM, 1000 J/m2), respectively, whereas no photochemical mutagenicity was observed with moxifloxacin (400 microM, 9000 J/m2). We suggest that the previously reported ability of lomefloxacin and Bay y3118 to photochemically produce oxidative DNA damage, which is known to be mutagenic, may be the basis for the photochemical mutagenicity and the reported photochemical carcinogenicity. The photostable moxifloxacin appears to lack such properties.

Mechanistic basis for nonlinearities and thresholds in rat liver carcinogenesis by the DNA-reactive carcinogens 2-acetylaminofluorene and diethylnitrosamine.

To explore differences in mechanisms of carcinogenicity at low and high exposures, we have conducted a series of exposure-response studies of hepatocarcinogenesis in rats using 2 well-studied DNA-reactive carcinogens, 2-acetylaminofluorene and diethylnitrosamine. In these studies, we have used intraperitoneal injection or intragastric instillation to deliver exact doses during an initiation segment followed by phenobarbital as a liver tumor promoter to enhance manifestation of initiation. This protocol results in carcinogenicity comparable to that produced by lifetime exposure to the carcinogens. Our findings in these experiments provide evidence for the following: (a) formation of DNA adducts can be nonlinear, with a plateau at higher exposures; (b) cytotoxicity shows no-effect levels and is related to exposure; (c) compensatory hepatocyte proliferation shows no-effect levels and can be supralinear at high exposures; (d) formation of preneoplastic hepatocellular altered foci can show no-effect levels and appears supralinear at high exposures; (e) no-effect levels can exist for tumor development, and the exposure response can be supralinear. We interpret these findings to reflect thresholds for hepatocellular initiating effects of these carcinogens and exaggerated responses at high exposures attributable to cytotoxicity and compensatory hepatocyte proliferation. Such enhanced proliferation of hepatocytes harboring DNA damage likely results in an exaggerated yield of mutations in critical genes, leading to supralinear initiation of carcinogenesis. Thus, mechanisms differ between low and high exposures. Based on these observations, we suggest that linear extrapolation from high toxic exposures to postulated low-exposure effects of DNA-reactive carcinogens can yield overestimates. Such extrapolation must be supported by mechanistic information. The finding of no-effect levels provides a basis for understanding why low-level environmental exposures of humans to even DNA-reactive carcinogens may convey no cancer risk.

Lack of DNA-damaging activity of five non-nutritive sweeteners in the rat hepatocyte/DNA repair assay.

The non-nutritive sweeteners acesulfame-K, aspartame, cyclamate, saccharin and sucralose were tested for DNA damaging activity in the rat hepatocyte/DNA repair assay. Using hepatocytes from F344 and Sprague-Dawley male rats, all were inactive despite strong responses for the positive control, 2-aminofluorene.

Oxidative DNA damage: endogenous and chemically induced.

A variety of types of DNA oxidation occur endogenously and mediated by xenobiotics. Certain forms are mutagenic and carcinogenic and may lead to other pathologies.

Does smoking reduction result in reduction of biomarkers associated with harm? A pilot study using a nicotine inhaler.

The aim of the study was to determine if smoking reduction using a nicotine inhaler in heavy cigarette smokers who wanted to reduce but not stop smoking results in decreased levels of known biomarkers of harm. The study design was a one-sample within-subject comparative open-label study of 23 (10 male and 13 female) subjects using a nicotine inhaler to reduce smoking, with follow-up at 24 weeks. A structured protocol was used with a smoking-reduction schedule from 40 or more cigarettes per day to 10 cigarettes per day by week 9. Behavioral counseling was provided by a research assistant and ad lib use of the nicotine inhaler for 12 weeks was permitted. Blood thiocyanate, cotinine, 4-aminobiphenyl hemoglobin adducts; urine NNAL and NNAL-glucuronide; and expired air carbon monoxide were measured. On average, the subjects were able to reduce their smoking by over 50% at week 12, but only two were able to reduce to 10 cigarettes per day. The reported reduction in smoking was not associated with a consistent reduction in the biomarkers. There was no reduction in the NNAL, 4-aminobiphenyl hemoglobin adducts nor carbon monoxide levels of expired air. There was a significant reduction of NNAL-glucuronide and the sum of NNAL and NNAL-glucuronide but only at week 24. Thiocyanate levels increased. Before widely promoting harm reduction as a treatment strategy for heavy smokers, more research needs to be performed to prove conclusively that such smokers who want to reduce but not stop can actually reduce and maintain their smoking rate at a level which is likely to reduce harm. It also needs to be determined whether a reduction in the smoking rate translates into reduction of harm. At the present, for heavy smokers, an abstinence approach seems to be more scientifically sound.

Diethylnitrosamine exposure-responses for DNA ethylation, hepatocellular proliferation, and initiation of carcinogenesis in rat liver display non-linearities and thresholds.

In previous exposure-response studies, we have documented non-linearities for some of the early effects in rat liver of diethylnitrosamine (DEN) and a near no-effect levels for initiation of promotable liver neoplasms at the lowest cumulative exposure of 0. 5 mmol/kg body weight; this in spite of formation of DNA adducts and induction of hepatocellular altered foci (HAF). To extend these investigations, in an initiation segment, young male F344 rats were administered four exposures of DEN ranging from a cumulative total of 0.25 mmol, which is half of the previously used low exposure, up to 2 mmol per kg body weight, an effective initiating exposure. These exposures were achieved by once weekly intragastric instillations of one-tenth the total exposures for up to 10 weeks. The initiation segment was followed by a 4 week recovery segment, to allow for remission of acute and subchronic effects of DEN, after which the groups were maintained on 0.06% phenobarbital in the diet for 24 weeks to promote liver tumor development in order to assess initiation. During and after initiation and at the end of recovery, selected groups were studied for several crucial effects involved in hepatocarcinogenicity. The low exposure produced a low-level of DNA ethylation at both 5 and 10 weeks of exposure, measured as O(4)-ethylthymidine, the most persistent promutagenic ethylation product. At the 5 week interval, the adduct values of the higher exposures were less than proportional to the increment of exposure, suggestive of nonlinearity. Assessment of cellular proliferation by staining for proliferating cell nuclear antigen revealed that the lowest exposure did not increase the replicating fraction of hepatocytes during the initiation (10 weeks) or recovery (4 weeks) segments, whereas in the three higher exposure groups, proliferation was increased in relation to dose and time. Preneoplastic HAF expressing glutathione S-transferase-placental-type were present at low multiplicity in control livers and their multiplicity was increased in all exposure groups by the end of exposure, at which time the increase in the high exposure group was disproportionately greater than the increment of exposure. After phenobarbital administration in the promotion segment, all exposure groups exhibited further HAF increases at 39 weeks. At the end of the promotion segment, no hepatocellular neoplasm was found in 80 controls or in 40 rats in the low exposure group. In the mid-low exposure group, which was the previously studied low exposure, only one adenoma was found, yielding a 3% incidence, while in the two higher exposure groups, 32 and 80% of rats exhibited liver neoplasms, which were increased disproportionately greater than the increments of exposure. Thus, the findings document non-linearities of early DEN effects and at the lowest cumulative dose, a no-effect level (NEL) or threshold for initiation of promotable liver neoplasms. These findings provide a conceptual basis for understanding why low-level exposures to DNA-reactive carcinogens may convey no cancer risk.

Nonlinearities in 2-acetylaminofluorene exposure responses for genotoxic and epigenetic effects leading to initiation of carcinogenesis in rat liver.

The dose responses for several effects of low-level limited exposures to 2-acetylaminofluorene (AAF) in the livers of male Fischer 344 rats were measured and a subsequent phenobarbital tumor promotion regimen was used to manifest initiation of carcinogenesis. Three doses over a 10-fold range yielding cumulative total exposures of 0.126, 0.42, and 1.26 mmol AAF/kg body weight were achieved by daily intragastric instillation for up to 12 weeks with interim terminations. This was followed by 24 weeks administration of 500 ppm phenobarbital (PB) in the diet to promote liver tumor development. At 12 weeks at the end of AAF administration, all exposures produced adducts in liver DNA, measured by 32P postlabeling, and the level of adducts increased with exposure, except that the high exposure did not produce a dose proportional increase. Measurement of arylsulfotransferase activity, a key enzyme in the metabolic activation of AAF, revealed that in livers from the high exposure animals, the enzyme was inhibited. To assess for toxicity, the centrilobular zone of glutamine synthetase-positive hepatocytes was quantified immunohistochemically at 12 weeks. The area of the zone was reduced in the high exposure group and there was a trend to reduction in relationship to exposure. The two lower exposures to AAF produced no increase in cell proliferation, whereas the high exposure resulted in a marked increase, about 8-fold over controls. Initiation was assessed by induction of hepatocellular altered foci (HAF) that expressed the placental form of glutathione S-transferase. AAF induced HAF in the high exposure group, 9-fold at 8 weeks and 170-fold at 12 weeks compared to controls. In rats maintained on PB for 24 weeks after exposure, the multiplicity of HAF increased in controls and comparably in the low and mid exposure groups, but remained at the about the same high level in the high exposure group. The high exposure produced a substantial incidence of benign neoplasms by 12 weeks, and with promotion by 36 weeks, all rats developed hepatocellular neoplasia. In the mid exposure group, only one adenoma occurred at 36 weeks in 17 rats, while in the low exposure group, no liver tumor occurred in 23 rats. Thus, these findings document nonlinearities for some of the effects of AAF, with supralinear effects at the high exposure for cell proliferation and induction of HAF, and a no-observed-effect level for induction of promotable liver neoplasms at the lowest cumulative exposure of 0.126 mmol/kg, in spite of the formation of DNA adducts. We conclude that the effects of this DNA-reactive hepatocarcinogen leading to initiation exhibit nonlinearities and possible thresholds.