David L. Busbee

Induction of Aryl Hydrocarbon Hydroxylase in Human Pulmonary Alveolar Macrophages by Cigarette Smoking

Abstract Pulmonary alveolar macrophages were obtained from healthy volunteers by saline pulmonary lavage, and aryl hydrocarbon hydroxylase was measured in the cells. Enzyme activity was low in cells from five nonsmokers with a mean of 0.008±0.004 U/106 cells. Cells obtained from nine cigarette smokers contained higher enzyme levels, with a mean of 0.095±0.024 U/106 cells. A former cigarette smoker was lavaged on five occasions. Enzyme activity during two lavages 4 mo apart were 0.010 and 0.009 U/106 cells, respectively. 1 wk after smoking was resumed, the enzyme activity rose slightly to 0.013, and reached 0.041 U/106 cells by 1 mo. Upon cessation of smoking, the enzyme activity returned to control levels by the next lavage, 2 mo later. These data indicate that aryl hydrocarbon hydroxylase may be induced in pulmonary alveolar macrophages of subjects chronically exposed to cigarette smoke.

Activation of a low specific activity form of DNA polymerase α by inositol-1,4-bisphosphate

A low activity form of DNA polymerase alpha immunoaffinity-purified from adult-derived human fibroblasts was activated by interaction with phosphatidylinositol-4-monophosphate, while a high activity form of the enzyme did not interact with phosphatidylinositol-4-monophosphate or its derivatives. Phosphatidylinositol-4-monophosphate was apparently hydrolyzed in the presence of a highly purified low activity form of DNA polymerase alpha, effecting the release of diacylglycerol and the retention of inositol-1,4-bisphosphate by the enzyme complex. The resulting inositol-1,4-bisphosphate/protein complex exhibited increased affinity of binding to DNA template/primer and increased deoxynucleotidyltransferase activity. These data indicate that inositol-1,4-bisphosphate may function as an effector molecule in the activation of a low activity form of human DNA polymerase alpha and suggest that it may function as a second messenger during the initiation of mitosis in stimulated cells.

Homologies between human and dolphin chromosomes detected by heterologous chromosome painting

Human chromosome-specific probes for the entire karyotype were hybridized to metaphase spreads of the Atlantic bottlenose dolphin, Tursiops truncatus, to directly compare the evolutionary conservation of chromosomal segments between these two distantly related species. All human chromosomal paints, except the Y probe, hybridized to Tursiops counterparts, and every dolphin chromosome was painted except for the smallest submetacentric pair. In our analysis, 36 segments of conserved synteny common to the human and dolphin genomes were identified. The distribution of conserved chromosomal segments and the specific rearrangement patterns found between the two genomes are presented and discussed.

Analysis of aryl hydrocarbon hydroxylase activity in human lung tissue, pulmonary macrophages, and blood lymphocytes.

Aryl hydrocarbon hydroxylase (AHH) activity was measured fluorometrically in surgically‐excised fresh lung tissue, pulmonary alveolar macrophages (PAMs), and peripheral blood lymphocytes from 14 cigarette smokers (7 with and 7 without primary lung cancer). Levels of AHH in fresh PAMs and AHH inducibility (expressed as fold‐induction) in cultured, mitogen‐stimulated lymphocytes from individual noncancer patients correlated well (r =0.975, p < 0.001). For individual lung cancer patients, however, these values were dissociated (linear regression not appropriate for this set of values). Levels of AHH in fresh lung tissue and fold‐induction ratios in cultured lymphocytes from individual noncancer patients also exhibited a positive correlation (r =0.976, p < 0.001), while values for individual lung cancer patients did not (r =0.007, p =0.987). A close agreement was noted for AHH in fresh lung tissue and fresh PAMs from individual noncancer patients (r =0.984, p < 0.001), while these values are weakly correlated for lung cancer patients (r =0.658, p < 0.11). When AHH activity in fresh PAMs, in fresh lung tissue, and AHH inducibility in cultured lymphocytes were simultaneously compared, an excellent relationship was observed for values for all 3 tissues for individual noncancer patients (r =0.987, p < 0.001). However, AHH levels in these 3 tissues from individual lung cancer patients were not correlated (r =0.701, p > 0.25). These results indicate similar capacity for AHH induction is present in fresh lung tissue, fresh PAMs, and cultured mitogen‐stimulated lymphocytes from cigarette smokers without evidence of lung cancer, but AHH values are not positively correlated with similar tissues from individual lung cancer patients.

Comparative uptake, vascular transport, and cellular internalization of aflatoxin-B1 and benzo(a)pyrene

Studies of the uptake of benzo(a)pyrene (BaP) and aflatoxin-B1 (AFB1) after gastric instillation showed that BaP was absorbed via the intestinal lymphatic drainage and transported to the vascular circulation sequestered within lipoproteins in thoracic duct lymph, while AFB1 was absorbed with water soluble compounds into the gastrointestinal venous drainage and was not transported in association with lipoproteins. BaP was taken up into plasma lipoproteins over a broad concentration range, while AFB1 was not sequestered within lipoproteins over the same concentration range. Low density lipoproteins (LDL) facilitated BaP uptake into fibroblasts and impeded BaP uptake into hepatocytes. High density lipoproteins (HDL) facilitated BaP uptake into hepatocytes and impeded BaP uptake into fibroblasts. The uptake of AFB1 into either fibroblasts or hepatocytes was not affected by lipoproteins.

DEHP, bis(2)-ethylhexyl phthalate, alters gene expression in human cells: possible correlation with initiation of fetal developmental abnormalities

Diethylhexylphthalate (DEHP) is a widely distributed phthalate, to which humans are exposed to due to its variety of commercial and manufacturing uses. As a plasticiser, it is found in a wide number of products, and metabolites of DEHP have been detected in urine samples from a high percentage ofthe peoplescreened for phthalates. We utilised DNA microarray analysis to evaluate DEHP for gene expression disrupting activity using the human cell line MCF-7, and found that DEHP significantly dysregulated approximately 34% of the 2400 genes spotted on the NEN2400 chip we used. The results suggest that DEHP, a known estrogen agonist and probable androgen antagonist, alters the expression of a number of genes, many of which are critical for fetal development. Down-regulation of two genes, FGD1 and PAFAH1B1, related in that both are essential for fetal brain development, was corroborated using quantitative real time PCR. These studies show DEHP to be a highly effective human gene expression-altering chemical, and that, at appropriate concentrations, it has the possibility of altering fetal central nervous system development, resulting in the birth defects lissencephaly and/or faciodigitogenital dysplasia.

Alteration of estrogen-regulated gene expression in human cells induced by the agricultural and horticultural herbicide glyphosate

Gene expression is altered in mammalian cells (MCF-7 cells), by exposure to a variety of chemicals that mimic steroid hormones or interact with endocrine receptors or their co-factors. Among those populations chronically exposed to these endocrine disruptive chemicals are persons, and their families, who are employed in agriculture or horticulture, or who use agricultural/horticultural chemicals. Among the chemicals most commonly used, both commercially and in the home, is the herbicide glyphosate. Although glyphosate is commonly considered to be relatively non-toxic, we utilized in vitro DNA microarray analysis of this chemical to evaluate its capacity to alter the expression of a variety of genes in human cells. We selected a group of genes, determined by DNA microarray analysis to be dysregulated, and used quantitative real-time PCR to corroborate their altered states of expression. We discussed the reported function of those genes, with emphasis on altered physiological states that are capable of initiating adverse health effects that might be anticipated if gene expression were significantly altered in either adults or embryos exposed in utero. Human & Experimental Toxicology (2007) 26, 747—752

Decreased fidelity of DNA polymerases and decreased DNA excision repair in aging mice: Effects of caloric restriction

Hepatic DNA polymerases from calorie restricted and ad libitum 26 month old C57BL/6 mice showed a decline in fidelity of nucleotide incorporation compared with weanling animals. Both alpha and beta polymerases from calorie restricted aged mice exhibited a higher level of fidelity than polymerases from ad libitum aged mice. UV-initiated unscheduled DNA synthesis was significantly higher in hepatocytes from weanling and 18 month old calorie restricted animals compared with cells from 18 month old ad libitum animals, while MMS-initiated unscheduled DNA synthesis did not differ significantly between cells from young and old or ad libitum and calorie restricted animals. These data suggest that calorie restriction could play a significant role in decreasing the age-related decline of cellular mechanisms expected to reduce the rate at which mutations accumulate during aging, and could potentially prolong the onset age of mutation-associated diseases of the elderly.

Uptake and vascular transport of ingested aflatoxin

The uptake and vascular transport of abomasally instilled aflatoxin B1 (AFB1) was investigated in sheep. Aflatoxin uptake was compared with that of palmitate, a water-insoluble oil known to be absorbed into the intestinal lymphatic drainage which bypasses the liver to enter the peripheral vascular circulation via the thoracic duct. After instillation into the abomasum, aflatoxin was detected in inferior vena cava blood within 30 min, while palmitate was not detected in vena cava blood at any time. Palmitate was detected in thoracic duct lymph after about 2 h. More than 95% of the palmitate in lymph was associated with the chylomicron fraction, while aflatoxin in either plasma or lymph was not detectably associated with any of the circulating lipoproteins. In addition, aflatoxin did not partition into plasma or lymph lipoproteins in vitro. Toxic lipophilic xenobiotics, such as benzo(a)pyrene and polychlorinated biphenyls (PCBs) do partition into lipoproteins, are absorbed into the intestinal lymphatic drainage, bypassing the liver to enter the peripheral vascular circulation directly, and are not specifically hepatotoxic. These data suggest that the mode of aflatoxin absorption from the gastrointestinal system results in its immediate transport to the liver, which may contribute to aflatoxin hepatotoxicity.

PCBs and chlorinated pesticides in clinically healthy Tursiops truncatus: relationships between levels in blubber and blood

A method for estimating organochlorine (OC) levels in blubber from levels found in blood samples could augment health assessment of dolphin populations where blubber sample collection is not possible. To explore the relationship between residue levels in red blood cells and blubber, paired preprandial blubber and blood samples were collected from 16 clinically healthy bottlenose dolphins, Tursiops truncatus. Residue levels were quantified for 10 PCB congeners and 17 chlorinated pesticides using GC/MS. Not all OCs detected in blubber were found in detectable levels in blood. However, significant relationships (p ≤ 0.05) were found for HCB, trans-nonachlor, o,p′-DDD, p,p′-DDD, o,p′-DDE, p,p′-DDE, o,p′-DDT, tDDT, and PCBs 52, 101, 118, 128, 138(+158), 153, 170, and 187. The statistically significant r2 terms ranged from 0.95 (p,p′-DDD) to 0.36 (trans-nonachlor) with a median of 0.64. The slopes of the regression models presented here for these analytes may be useful in estimating population levels of OCs in Tursiops blubber using levels measured in blood samples.