Cheryl A. Marietta

Effects of ethanol administration on parameters of immunocompetency in rats.

Ethanol administered to rats intragastrically in doses sufficient to cause dependency resulted in a rapid cell loss from the thymus and spleen. Cell loss from the peripheral blood was due primarily to a loss of lymphocytes, but a concomitant granulocytosis resulted in only small changes in the total leukocyte count. Lymphocyte proliferation to both T‐ and B‐cell mitogens was severely compromised by ethanol treatment. The cell loss and functional lymphocyte impairment also occurred at half the ethanol dose required to induce dependency. Although cell numbers recovered relatively quickly after ethanol withdrawal, lymphocyte function, as measured by proliferation, recovered more slowly. Ethanol administration before or during immunization with sheep erythrocytes resulted in an impairment in the ability of animals to respond with a primary immune response to this antigen. These data suggest that ethanol given in quantities sufficient to produce dependence impairs in vitro and in vivo parameters of immunocompetency.

Transcriptional bypass of bulky DNA lesions causes new mutant RNA transcripts in human cells

Here, we characterize the mutant transcripts resulting from bypass of an 8,5′‐cyclo‐2′‐deoxyadenosine (cyclo‐dA) or cyclobutane pyrimidine dimer (CPD) by human RNA polymerase II (Pol II) in vivo. With the cyclo‐dA lesion, we observed two new types of mutant transcripts. In the first type, the polymerase inserted uridine opposite the lesion and then misincorporated adenosine opposite the template deoxyadenosine downstream (5′) of the lesion. The second type contained deletions of 7, 13 or 21 nucleotides (nt) after uridine incorporation opposite the lesion. The frequency of the different types of transcript from the cyclo‐dA lesion in mutant human cell lines suggests that the Cockayne syndrome B protein affects the probability of deletion transcript formation. With the CPD‐containing construct, we also detected rare transcripts containing 12 nt deletions. These results indicate that RNA pol II in living human cells can bypass helix‐distorting DNA lesions that are substrates for nucleotide excision repair, resulting in transcriptional mutagenesis.

Acute ethanol administration selectively alters localized cerebral glucose metabolism

The effects of acute ethanol administration on glucose utilization in the CNS of rat were studied using the 2-deoxyglucose technique. Cerebral glucose utilization was determined for 53 brain regions at peak and descending blood ethanol concentrations averaging 14, 26 and 66 mM. Decreased glucose utilization was the predominant finding and was observed in 20% of the regions evaluated, with median raphe, vestibular nucleus, cerebellar vermis, and various structures associated with the auditory system showing the greatest reductions. The only structures that showed increased glucose utilization were the dentate region of the hippocampus and the superior olive, and this was only apparent at a blood ethanol concentration of 14 mM.

Effect of adrenalectomy on ethanol-associated immunosuppression

The alterations in lymphoid cell numbers and lymphocyte function due to administration of ethanol was found to be associated with high levels of circulating corticosteroids. The role of corticosteroids in the ethanol-induced alterations in the immune system was studied by administering ethanol to adrenalectomized rats. The results of these experiments showed that the ethanol-induced loss of cells from the thymus was not completely prevented by adrenalectomy and the ethanol-induced loss of cells from the spleen was not affected by adrenalectomy. Likewise the ethanol-induced decrease in antibody production to the T-cell-dependent antigen sheep erythrocytes were not affected by adrenalectomy. The ability of animals to produce antibodies of the T-cell-independent antigen, TNP-Ficoll, was not affected by ethanol regardless of whether the animals had adrenal glands or not. These data indicate that adrenal corticosteroids are responsible for some but not all of the thymic involution due to ethanol intoxication. Also, adrenalectomized rats did not show as much impairment in lymphocyte proliferation as sham adrenalectomized animals after ethanol administration. However, this loss of cells from peripheral lymphoid organs such as the spleen and the decreased ability to respond to T-cell-dependent antigens is not influenced by adrenalectomy indicating mechanisms other than corticosteroids mediate these effects of ethanol.

Cerebral 2-deoxyglucose uptake in rats during ethanol withdrawal and postwithdrawal.

The overt ethanol withdrawal syndrome is associated with a generalized increase in cerebral uptake of 2-deoxyglucose. Relatively high elevations of 2-deoxyglucose were observed in many structures associated with motor function, the mamillary body-anterior thalamus-cingulate cortex pathway, many thalamic nuclei, and the raphe. Overtly withdrawing rats had higher levels of 2-deoxyglucose than postwithdrawing animals that had been abstinent for 1-5 weeks in 96% of the gray areas evaluated. Postwithdrawal was associated with increased amounts of 2-deoxyglucose in comparison to controls in 80% of the gray areas evaluated. Postwithdrawal and control rats did not differ in some areas involved with motor function and some limbic structures, such as the mamillary body-anterior thalamus-cingulate cortex pathway. It is concluded that the ethanol-withdrawal syndrome results in alterations in cerebral physiology, some of which persist for at least 5 weeks postwithdrawal.

Acetaldehyde stimulates FANCD2 monoubiquitination, H2AX phosphorylation, and BRCA1 phosphorylation in human cells in vitro: implications for alcohol-related carcinogenesis.

According to a recent IARC Working Group report, alcohol consumption is causally related to an increased risk of cancer of the upper aerodigestive tract, liver, colorectum, and female breast [R. Baan, K. Straif, Y. Grosse, B. Secretan, F. El Ghissassi, V. Bouvard, A. Altieri, V. Cogliano, Carcinogenicity of alcoholic beverages, Lancet Oncol. 8 (2007) 292-293]. Several lines of evidence indicate that acetaldehyde (AA), the first product of alcohol metabolism, plays a very important role in alcohol-related carcinogenesis, particularly in the esophagus. We previously proposed a model for alcohol-related carcinogenesis in which AA, generated from alcohol metabolism, reacts in cells to generate DNA lesions that form interstrand crosslinks (ICLs) [J.A. Theruvathu, P. Jaruga, R.G. Nath, M. Dizdaroglu, P.J. Brooks, Polyamines stimulate the formation of mutagenic 1,N2-propanodeoxyguanosine adducts from acetaldehyde, Nucleic Acids Res. 33 (2005) 3513-3520]. Since the Fanconi anemia-breast cancer associated (FANC-BRCA) DNA damage response network plays a crucial role in protecting cells against ICLs, in the present work we tested this hypothesis by exposing cells to AA and monitoring activation of this network. We found that AA exposure results in a concentration-dependent increase in FANCD2 monoubiquitination, which is dependent upon the FANC core complex. AA also stimulated BRCA1 phosphorylation at Ser1524 and increased the level of gammaH2AX, with both modifications occurring in a dose-dependent manner. However, AA did not detectably increase the levels of hyperphosphorylated RPA34, a marker of single-stranded DNA exposure at replication forks. These results provide the initial description of the AA-DNA damage response, which is qualitatively similar to the cellular response to mitomycin C, a known DNA crosslinking agent. We discuss the mechanistic implications of these results, as well as their possible relationship to alcohol-related carcinogenesis in different human tissues.

Expression of long-patch and short-patch DNA mismatch repair proteins in the embryonic and adult mammalian brain

Expression of the DNA mismatch repair (MMR) pathway was examined in the adult and developing rat brain. Rat homologues of human GTBP and MSH2, which are essential components of the post-replicative DNA MMR system, were identified in nuclear extracts from the adult and developing rat brain. Developmental studies showed that both GTBP and MSH2 levels were higher in nuclei isolated from the embryonic brain (day 16) than adult brain. However, this difference was not as dramatic as the difference in the number of proliferating cells. Levels of thymine DNA glycosylase (TDG), the enzyme which catalyzes the first step in short patch G:T mismatch repair, were also decreased in adult compared to embryonic brain. In the adult brain, MMR proteins were elevated in nuclear extracts enriched for neuronal nuclei. These results suggest that adult brain cells have the capacity to carry out DNA mismatch repair, in spite of a lack of ongoing DNA replication.

Ethanol-withdrawal syndrome associated with both general and localized increases in glucose uptake in rat brain

Glucose uptake was studied in the brains of rats undergoing an overt ethanol-withdrawal syndrome by 2-deoxy-D-[14C]glucose autoradiography. In addition to a general increase in glucose uptake, localized alterations were observed in sensorimotor cortex, globus pallidus, thalamus and cerebellum. The results suggest that the ethanol-withdrawal syndrome is associated with a general increase in glucose metabolism as well as localized increases in functionally distinct regions of sensory and motor brain regions.

Ethanol dependence and withdrawal selectively alter localized cerebral glucose utilization

The 2-deoxyglucose technique was used to determine local cerebral glucose utilization (LCGU) in over 50 brain regions of rats physically dependent upon ethanol and compared to those of acutely intoxicated and those undergoing an overt ethanol-withdrawal syndrome. Dependent-intoxicated rats (average blood ethanol concentration 64 mM) had decreased LCGU in 13/54 regions, including those associated with the limbic system, cerebellum, and motor system. The ethanol withdrawal syndrome was associated with 17/50 gray regions showing an increase, including regions involved with motor function, auditory system, and mammillary bodies-anterior thalamus-cingulate cortex pathway. The most pronounced differences between these groups occurred in regions associated with motor function, cerebellar function, anterior thalamus, and median raphe. Comparisons between dependent-intoxicated and acutely intoxicated rats (average blood ethanol concentration 66 mM) revealed that acute intoxication was associated with a relatively greater reduction in LCGU in regions involved with sensory-related functions, mammillary bodies, and median raphe. With the development of dependence, adaptation occurred in these regions except for inferior colliculus and median raphe. Dependence was also associated with a relative decrease in LCGU in white matter, limbic system, and extrapyramidal motor system.

Cerebral glucose utilization in rat brain during phenobarbital withdrawal

The phenobarbital withdrawal syndrome in rats is characterized by tremors, arched back, weight loss and hyperactivity. This syndrome is shown to be associated with both general and localized increases in cerebral glucose utilization. An increase in glucose utilization (significant at the P less than or equal to 0.001 level) was observed in 72% of the 57 structures examined. Increases in glucose utilization of greater than or equal to 180% of control values were noted in structures associated with the motor system (columns in the frontal sensorimotor cortex, globus pallidus, dentate nucleus of the cerebellum and ovoid areas in the cerebellar vermis), thalamic nuclei (lateral and posterior), dorsal lateral geniculate, mammillary body, cingulate cortex, locus ceruleus, and cerebellar flocculus and paraflocculus. The structures showing the greatest increase in glucose utilization were cerebellar paraflocculus (257% of control), columns in the frontal sensorimotor cortex (247% of control) and ovoid areas in the cerebellar vermis (223% of control). Areas of the brain that have been described as cell body areas for serotonergic (raphe), noradrenergic (locus ceruleus), dopaminergic (substantia nigra, zona compacta) and GABAergic (globus pallidus) neurons also showed increases in glucose utilization. The pattern of cerebral glucose utilization accompanying the phenobarbital withdrawal syndrome in rats contrasts with that for morphine withdrawal and exhibits both similarities and differences with respect to ethanol withdrawal.