Effects of developmental exposure to mixtures of environmental contaminants on the hepatic metabolism of estradiol-17ß in immature female Sprague Dawley rats.

Exposure to environmental contaminants induces the activation of cytochrome P450s (CYP) which lead to the hydroxylation of contaminants and endogenous hormones such as estrogens. The hydroxylation of estrogens forms catecholestrogens (CEs), one of them being the mutagenic 4-hydroxyestradiol-17ß (4-OH-E2). Catecholestrogens are transformed by catechol-o-methyltransferases (COMTs) into nonreactive methoxyestrogens. To investigate the hepatic metabolism of estradiol-17ß in female offspring at postnatal day (PND) 21, pregnant rats were dosed daily from gestation day 1 until PND 21 with 2 dose levels of organochlorine pesticides (OCPs; 0.019 or 1.9 mg/kg per d), methylmercury (MeHg; 0.02 or 2 mg/kg per d), polychlorinated biphenyls (PCBs; 0.011 or 1.1 mg/kg per d), or a mixture (M; 0.05 or 5 mg/kg per d) including all 3 groups of chemicals. Concentrations of organochlorines in the mixture M were based on their proportions in serum of the Canadian Arctic population. The messenger RNA (mRNA) expressions of CYP and COMT were analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). High-performance thin layer chromatography and phosphor imaging were used to measure the transformation of (14)C substrates into estrogen metabolites. The low-dose treatments or the MeHg groups had no effect. The high-dose OCP, PCB, and M group increased the production of 2-OH-E2 and 6α-OH-E2, while only the PCB and M groups increased the 2-OH-CE/methoxyestrogen ratio. In all groups, the cytosolic COMT activity exceeded the microsomal production rate of 4-OH-E2. Although the M treatment included the PCB and OCP mixtures, it did not modify the estrogen metabolism more than did the PCB mixture alone. This endocrine disruption information contributes to our understanding of chemical interactions in the toxicology of chemical mixtures.

Extending the transposable payload limit of Sleeping Beauty (SB) using the Herpes Simplex Virus (HSV)/SB amplicon-vector platform.

The ability of a viral vector to safely deliver and stably integrate large transgene units (transgenons), which not only include one or several therapeutic genes, but also requisite native transcriptional regulatory elements, would be of significant benefit for diseases presently refractory to available technologies. The herpes simplex virus type-1 (HSV-1) amplicon vector has the largest known payload capacity of approximately 130 kb, but its episomal maintenance within the transduced cell nucleus and induction of host cell silencing mechanisms limits the duration of the delivered therapeutic gene(s). Our laboratory developed an integration-competent version of the HSV-1 amplicon by adaptation of the Sleeping Beauty (SB) transposon system, which significantly extends transgene expression in vivo. The maximum size limit of the amplicon-vectored transposable element remains unknown, but previously published plasmid-centric studies have established that DNA segments longer than 6-kb are inefficiently transposed. Here, we compared the transposition efficiency of SB transposase in the context of both the HSV amplicon vector as well as the HSV amplicon plasmid harboring 7 and 12-kb transposable reporter transgene units. Our results indicate that the transposition efficiency of the 12-kb transposable unit via SB transposase was significantly reduced as compared with the 7-kb transposable unit when the plasmid version of the HSV amplicon was used. However, the packaged HSV amplicon vector form provided a more amenable platform from which the 12-kb transposable unit was mobilized at efficiency similar to that of the 7-kb transposable unit via the SB transposase. Overall, our results indicate that SB is competent in stably integrating transgenon units of at least 12 kb in size within the human genome upon delivery of the platform via HSV amplicons.

Regulation of neuronal traits by a novel transcriptional complex.

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

Gene expression profiling in rat cerebellum following in utero and lactational exposure to mixtures of methylmercury, polychlorinated biphenyls and organochlorine pesticides.

Although human populations are continuously exposed to complex mixtures of contaminants, the effects of such exposure on the developing brain transcriptome are poorly characterized. Rats were exposed perinatally to the northern contaminant mixture (NCM) which was designed to reflect the blood contaminant profile of Canadian arctic populations, to components of the NCM administered separately (methylmercury (MeHg), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCs)) or to the goitrogen propylthiouracyl. Post-natal day (PND) 14 cerebellum global gene expression resulting from such exposures was investigated using high-density cDNA microarrays. Fifty known genes were identified as differentially expressed between the control group and at least one other treatment group. The microarray data were validated by quantitative PCR (qPCR) on a subset of 10 genes. The differentially expressed genes are involved in a variety of processes, including nerve cell differentiation, migration, myelination and synaptic transmission. The comparison of cerebellum gene expression profiles resulting from exposure to the NCM and its individual components in male and female pups revealed that (i) gender is a crucial biological variable influencing genomic response to environmental contaminants and (ii) contaminant co-exposure significantly masks the effects of individual mixture components on cerebellum gene expression.

Transcriptional regulation of ALV bursal lymphomagenesis.

Avian leukosis virus (ALV) induces bursal lymphoma in chickens after rare integration of proviral long terminal repeat (LTR) sequences next to the c-myc proto-oncogene. Labile LTR-binding proteins are essential for c-myc hyperexpression, since LTR-enhanced transcription, as well as the activity of the LTR-binding proteins, is specifically decreased following protein synthesis inhibition. This lability is restricted to hematopoetic cells of ALV-susceptible chicken strains, suggesting that the labile proteins play an important role in susceptibility to lymphomagenesis. Five proteins that interact with the ALV LTR enhancer were identified from bursal lymphoma cells: two proteins are stable (b and c), while three proteins (a1, a3, and b*) are labile in pre-B cell types. Two cDNAs (a1/EBP and a3/EBP) encoding leucine zipper proteins that bind to the ALV LTR enhancer were cloned using a lambda gt11 cDNA expression library made from bursal lymphoma cells. We are presently studying the roles of a1/EBP and a3/EBP in labile regulation of LTR-enhanced c-myc transcription and susceptibility to bursal lymphomagenesis.

Behavioral and thyroid effects of in utero and lactational exposure of Sprague-Dawley rats to the polybrominated diphenyl ether mixture DE71.

Exposure of rodents during gestation and lactation to polybrominated diphenyl ethers (PBDEs) has been reported to disrupt neurobehavioral function in offspring, as well as to disrupt thyroid function. To assess this we evaluated development and behavior after gestational and lactational exposure to the technical PBDE mixture DE71. Pregnant Sprague-Dawley rats were exposed to 0, 0.3, 3.0 or 30 mg/kg/day of DE71 from gestation day 1 to postnatal day (PND) 21 and were assessed on a wide range of behavioral functions from early postnatal period until old age (PND 450). DE71 exposure decreased thyroid hormone levels (T3 and T4) in mothers and offspring with offspring being more sensitive that mothers. Developmental landmarks, neuromotor function, anxiety, learning and memory were not affected by DE71 at any age. DE71 produced small changes in motor activity rearing only at PND 110 but not at any other age and no other activity measure was altered by DE71. Cholinergic sensitivity measured by nicotine-stimulated motor activity was not affected by perinatal DE71 exposure. Acoustic startle responses were potentiated by DE71 at PND 90 indicating delayed effects on sensory reactivity. Habituation was measured in motor activity tests at five ages but was not altered by DE71 at any age. Habituation measured in startle tests was also not affected by exposure to DE71. For thyroid hormone levels at PND 21, the lowest adverse effect level was 3.0 mg/kg. Few behavioral effects were observed and the lowest adverse effect level was 30 mg/kg. Our results confirm that DE71 produces transient effects on thyroid hormone levels but does not result in learning or motor impairment and does not alter non-associative learning (habituation).

Enhanced learning in mice parallels vector-mediated nerve growth factor expression in hippocampus.

Spatial learning requires the integrity of the nerve growth factor (NGF)-responsive septohippocampal pathway. Loss of a single NGF allele at the mouse NGF locus (heterozygous null, ngf(+/-)) reduces septohippocampal NGF levels and NGF-regulated cholinergic neurotransmitter enzymes and results in spatial learning deficits in adult animals. A herpes simplex virus (HSV) amplicon vector was utilized to locally deliver NGF to the hippocampus of mice heterozygous and wild type (ngf(+/+)) at the NGF gene locus. NGF gene transfer produced transient increases in NGF protein levels and choline acetyltransferase activity in both ngf(+/-) and ngf(+/+) mice. However, spatial learning capability was improved only in ngf(+/-) mice. In aggregate, these findings suggest that amplicon-directed expression of NGF in subjects with baseline septohippocampal dysfunction can correct spatial learning deficits.

Dendritic cells transduced with HSV-1 amplicons expressing prostate-specific antigen generate antitumor immunity in mice.

There is currently much interest in generating cytotoxic T lymphocyte (CTL) responses against tumor antigens as a therapy for cancer. This work describes a novel gene transfer technique utilizing dendritic cells (DCs), an extremely potent form of antigen-presenting cell (APC), and herpes simplex virus-1 (HSV-1) amplicons. HSV-1 amplicons are plasmid-based viral vectors that are packaged into HSV-1 capsids, but lack viral coding sequences. Amplicon vectors have been constructed that encode the model tumor antigen ovalbumin (HSV-OVA) and human prostate-specific antigen (HSV-PSA), a protein that is expressed specifically in prostate epithelium and prostate carcinoma cells. These amplicons were packaged using a helper virus-free system that produces vector stocks that are devoid of contaminating cytotoxic helper virus. Transduction of DCs with HSV-OVA or HSV-PSA and co-culture with CTL hybridomas results in specific activation, indicating that transduced DCs express these transgenes and process the tumor antigens for class I MHC presentation to CTL. Mice immunized with HSV-PSA-transduced DCs generate a specific CTL response that can be detected in vitro by a (51)Cr-release assay and are protected from challenge with tumors that express PSA. These results indicate that DCs transduced with HSV-1 amplicon vectors may provide a tool for investigation of the biology of CTL activation by DCs and a new modality for immunotherapy of cancer.

The avian C/EBPgamma gene encodes a highly conserved leucine zipper transcription factor.

The C/EBP family of transcription factors regulates viral and cellular CCAAT/enhancer element-mediated transcription. We report the isolation and characterization of genomic and cDNA clones encoding avian CCAAT/enhancer-binding protein-gamma (C/EBP gamma). A partial cDNA clone for a C/EBP-related gene was previously identified by expression library screening for proteins binding the A1 CCAAT/enhancer motif from the avian leukosis virus long terminal repeat [W. Bowers and A. Ruddell (1992) J. Virol. 66, 6578-6586]. Additional cDNA and genomic clones were generated and sequenced to identify the complete protein coding sequence of this gene. Sequence analysis indicates that this gene encodes the avian homolog of C/EBP gamma. As with the murine C/EBP gamma homolog, the avian C/EBP gamma gene is comprised of two exons, with the open reading frame encoded in exon 2. The 150-aa C/EBP gamma protein is highly conserved, as the avian protein shows more than 80% identity with the murine and human homologs. The sequence of the initiation methionine (-3 caaAUGa +4) from the 150-aa open reading frame has a non-optimal Kozak initiation sequence. In vitro transcription and translation assay of this avian cDNA followed by radioimmunoprecipitation assay using a murine C/EBP gamma antiserum indicates that this non-optimal initiation codon is used to express a 22-kDa DNA-binding protein.

HSV vector-mediated gene delivery to the central nervous system.

The efficient and targeted transfer of genes is the goal of gene therapy. In the central nervous system (CNS), this is challenging due in part to the exquisite anatomy of the brain. Herpes simplex virus (HSV) vectors are particularly amenable to CNS therapies as they are capable of transducing a variety of cells, have a large transgene capacity and can exist as either oncolytic or non-immunogenic vectors. The versatility of this vector platform and its potential molecular therapeutic use in two CNS disorders, Alzheimer's disease and malignant brain tumors, will be discussed.

Time-dependent exacerbation of amphetamine-induced taste aversions following exposure to footshock.

Previous studies have shown that stressors attenuate LiCI-induced conditioned taste aversions (CTA) but not morphine-induced CTA. The current studies examined the effects of footshock on the acquisition and extinction of amphetamine-induced CTA. Experiment 1 demonstrated that exposure to 30 footshocks between saccharin consumption and amphetamine injections did not alter either the acquisition or the extinction of amphetamine-CTA. Experiment 2 demonstrated that exposure to the same shock parameters 2 and 4 days before saccharin-amphetamine pairing increased the magnitude of amphetamine-CTA after one saccharin-amphetamine pairing and delayed the recovery from the CTA. Experiment 2 also demonstrated that footshock increased the initial neophobic response to novel saccharin but did not alter subsequent saccharin consumption among saline-injected animals. These results indicate that stress-induced facilitation of amphetamine CTA are time-dependent and contrast with reports that stressors attenuate LiCI CTA. They also add support to the contention that CTAs induced by self-administered drugs like amphetamine are qualitatively different from CTAs induced by toxic substances like LiCL.

Evaluation of stressor effects on intracranial self-stimulation from the nucleus accumbens and the substantia nigra in a current intensity paradigm.

The effect of uncontrollable footshock was evaluated in animals responding for intracranial self-stimulation from the nucleus accumbens and the substantia nigra (pars compacta) in a descending current intensity paradigm. Responding for brain stimulation from the nucleus accumbens was found to be affected by the stressor at the upper end of the rate-intensity curve. In contrast, responding for brain stimulation from the substantia nigra was unaffected by the stressor at any of the current intensities employed. The variations of responding for self-stimulation from the nucleus accumbens were unrelated to alterations of locomotor activity or rearing. It is suggested that stressor-provoked reductions of responding for intracranial self-stimulation are not a result of the brain stimulation taking on aversive properties, but rather reflect a reduction in the reinforcing or motivational value associated with the stimulation.

Region-specific reductions of intracranial self-stimulation after uncontrollable stress: possible effects on reward processes.

Rates of responding for intracranial self-stimulation from the medial forebrain bundle, nucleus accumbens and substantia nigra were evaluated in mice that had been exposed to either escapable shock, yoked inescapable shock or no shock treatment. Whereas performance was unaffected by escapable shock, marked reductions of responding from the medial forebrain bundle and nucleus accumbens were evident following the uncontrollable shock treatment. Responding from the substantia nigra was unaffected by the stress treatment. Uncontrollable shock is thought to reduce the rewarding value of responding for electrical brain stimulation from those brain regions in which stressors are known to influence dopamine activity.

Prevention of stressor-induced disturbances of self-stimulation by desmethylimipramine.

Following exposure to uncontrollable footshock mice displayed a pronounced reduction of responding for electrical brain stimulation from the nucleus accumbens. In mice that received repeated treatment with the tricyclic antidepressant, desmethylimipramine, the shock induced reduction of responding was attenuated. It was suggested that, among other things, uncontrollable stressors result in disturbances of motivational/reward processes, which are antagonized by repeated treatment with desmethylimipramine.

Responding for brain stimulation: stress and desmethylimipramine.

Stressors influence the activity of biogenic amines and provoke a variety of behavioral disturbances which have been considered as models of human depression. To evaluate the effects of stressors on reward processes, responding for electrical brain stimulation was assessed after acute or chronic shock, and the modification of performance by desmethylimipramine was determined. While escapable shock did not affect performance, inescapable shock reduced responding from the nucleus accumbens and medial forebrain bundle, but not from the substantia nigra. These deficits were were antagonized by repeated stressor application or by desmethylimipramine. Uncontrollable stressors may influence motivational processes subserved by some brain regions, and may thus influence affective state. Chronic stress or desmethylimipramine may induce adaptive neurochemical changes, thereby preventing the behavioral disturbances otherwise produced by stressors.

Stress enhances the response to reward reduction but not food-motivated responding.

The effect of a single exposure to foot shock stress on runway responding for food reinforcement was assessed in animals trained and tested with the same or changed reinforcement magnitude. Foot shock (30 1-s shocks, 1.0 mA) exerted no impact on runway responding in animals trained and tested with the same level of reinforcement magnitude regardless of the absolute level of reinforcement magnitude (i.e., either 15 pellets or 1 pellet). Similarly, foot shock exerted no impact on runway responding in animals trained with a small magnitude of reinforcement but tested with an increased magnitude of reinforcement. In contrast, foot shock enhanced the increase in runway latencies produced by a reduction in reinforcement magnitude. Because reductions in reinforcement magnitude are known to be aversive for animals, these data indicate that foot shock stress can alter the behavioral response to an aversive stimulus without disrupting behavioral responding for an appetitive reinforcer. They also suggest that stressor-induced alterations in appetitively motivated behaviors may be secondary to alterations in sensitivity to subtle aversive stimuli rather than by directly altering appetitive motivation.

The role of ethanol availability on stress-induced increases in ethanol consumption.

Exposure to stressors can increase ethanol consumption and ethanol can attenuate the behavioral and biochemical effects of stressors. This study determined whether the availability of ethanol during the period of exposure to repeated restraint alters the poststress increase in ethanol intake. Seven days of restraint increased ethanol intake on the first poststress test only in animals deprived of ethanol during the restraint period. These results indicate that the availability of ethanol during exposure to restraint can attenuate the impact of restraint on ethanol intake. Ethanol intake was also positively related to novelty-induced locomotion and restraint eliminated this relationship. However, amphetamine-induced locomotion was not altered by either restraint or EtOH intake. These results indicate that voluntary ethanol intake can attenuate the impact of restraint stress and that restraint stress can alter the influence of ethanol on novelty-induced locomotion. It is suggested that this symmetrical relationship between ethanol intake and restraint stress may be involved in an interactive manner that determines stress-induced ethanol consumption.

How did Tarasoff affect clinical practice?

The Tarasoff decisions of the California Supreme Court in 1974 and 1976 held that psychotherapists could be held liable for failing to protect the victims of their potentially violent patients. Our survey of psychiatrists, psychologists, and social workers in eight metropolitan areas showed that Californians were more likely to have heard of the case, to believe it required warning the likely victim, and actually to issue warnings in such cases than were psychotherapists from other jurisdictions. Therapists were more willing to take steps to protect victims in 1980 than in 1975, but willingness to warn increased more among Californians than among those in other states. We conclude that although Tarasoff has influenced therapists' attitudes and behavior more in California than elsewhere, the case has also affected psychotherapeutic practice nationally.

Viral vector-induced expression of bone morphogenetic protein 2 produces inhibition of tumor growth and bone differentiation of stem cells.

A number of hormonal factors have been shown to be instrumental in the calcification process. This study represents an attempt at using one of these factors to specifically induce calcification of tumors to arrest tumor growth. The gene encoding bone morphogenetic protein 2 (BMP-2) was placed under transcriptional control of the promoter for carcinoembryonic antigen (CEA). This gene cassette was cloned into a herpes simplex virus (HSV) amplicon vector (HSV-CEA-BMP2). This vector was used to induce local BMP-2 production in CEA-expressing tumor cells to retard cell growth. Lysates of tumor cells treated with HSV-CEA-BMP2 were applied to stem cells to determine if BMP-2 expression promotes differentiation to bone lineage. pHSV-CEA-BMP2 efficiently transduced both CEA-expressing and non-expressing cells. BMP-2 was only expressed in CEA-positive cells. BMP-2 expression led to an inhibition of tumor cell growth. BMP-2 released by these CEA-expressing tumors also drove the differentiation of mesenchymal stem cells to bone lineage. This proof-of-concept study demonstrates that tumor cells can be specifically engineered to produce BMP-2, which leads to growth retardation and to the differentiation of non-committed stem cells to bone. This 'Medusa' effect can theoretically be exploited to retard tumor growth.