Cigarette Smoke-Induced Oxidative Stress and Autophagy in Human Alveolar Epithelial Cell Line (A549 Cells).

Chronic exposure to cigarette smoke (CS) causes structural and functional changes in the respiratory tract. It is a major risk factor for cardiovascular and systemic pulmonary diseases. The aim of this study was to investigate the effect of acute CS exposure (2 h) on oxidative stress, heat shock protein 70 (HSP70) expression, autophagy (LC3 expression), and oxidative stress (DCF fluorescence) in human alveolar epithelial cell line A549. Cell culture medium was conditioned with CS using commercial cigarettes, and A549 cells were grown in modified media for 2 h. In some experiments, A549 cells were pretreated with 100 µM of L-buthionine-sulfoximine (BSO) for 24 h to induce glutathione (GSH) depletion. In the cells grown in CS-conditioned medium, GSH was depleted by more than 30%, and reactive oxygen species were increased. Moreover, there was a considerable overexpression of HSP70 and a substantial accumulation of LC3. Similar changes were found when the cells were pretreated with BSO. We conclude that the short-term exposure of epithelial cells to CS increases oxidative stress that entails enhanced autophagy activity.

Cytotoxicity, Oxidative Stress, and Autophagy in Human Alveolar Epithelial Cell Line (A549 Cells) Exposed to Standardized Urban Dust.

Exposure to urban airborne particulate matter (PM) associates with adverse health effects, but the exact mechanisms remain unclear. In this study, we focused on cytotoxicity (MTT), oxidative stress (DCF/FC), DNA damage (PI/FC), necrosis/apoptosis (FC), and autophagy (LC3 expression; WB/FC) triggered by urban dust (UD) in naïve human alveolar epithelial A549 cells and in the cells with reduced glutathione (GSH). The A549 cells were grown in F12K/FCS media supplemented with coarse carbon black (CB; Huber990; 260 nm diameter; 200 µg·ml-1) or urban dust (UD; Standard Reference Materials; 200 µg·ml-1) for 24 h. To deplete intracellular glutathione (GSH), l-buthionine-(S,R)-sulfoximine (BSO; 100 mM; 24 h) was used. Pre-treatment with BSO depleted the cellular GSH by about 30%. A similar effect was noticed after UD. The CB was without any effects on the parameters tested, except for LC3 expression (autophagy) which increased by about twofold. However, UD decreased cell viability by about 27%, decreased cell proliferation in BSO pre-treated cells, increased ROS production, and increased both Hsp70 and LC3 proteins by about twofold, but most changes were unrelated to ROS-mediated GSH depletion. We conclude that urban dust-induced oxidative stress is important in PM toxicity, but other as yet unrecognized mechanisms are also involved.

Genetic regulation of mup production in recombinant inbred mice.

Inbred strains of mice excrete all three major urinary proteins (mups) when induced by testosterone, but differ as to the relative proportions and total levels of each mup present. We have now determined the urinary mup phenotypes before and after testosterone treatment of seven recombinant inbred strains derived from progenitor strains exhibiting different mup phenotypes. The results confirm previous observations indicating that total control of mup protein production is a multigenic process. One locus, Mup-a on chromosome 4, determines the relative mup protein proportions after induction by testosterone. Mup-a, together with other genetic sites, determines the basal mup proportions. Genes other than Mup-a determine the kinetics of mup induction and total mup excretion.

Regulation of mouse major urinary protein production by the Mup-A gene.

A method was developed to quantitate the daily excretion of the three major urinary proteins (mups) to test which parameters of the mup phenotype are controlled by the the Mup-a gene. Electrophoretic separation of the mup proteins, followed by staining and spectrophotometric scanning was used to characterize the phenotypes of various inbred strains. The mup phenotype of a strain proved to have two components: the absolute levels and the relative proportions of the mups present in the urine. Testosterone treatment alters both components of the mup phenotype, increasing mup excretion and aftering their relative proportions. The induced proteins are the same as the basal proteins as judged by electrophoretic mobility, molecular weight, and reactivity with antibody. All strains excrete all three mups when induced. The Mup-a gene appears to be a single, codominantly expressed regulatory locus that controls the induced proportions of the three proteins. However, other genes in addition to Mup-a participate in controlling the basal mup proportions, as well as individual and total mup levels before and after testosterone treatment.

Drug interactions with oral contraceptives: compilation and analysis of an adverse experience report database.

Over 700 alleged OC/drug interactions were reported for antituberculous drugs, other antibiotics, anticonvulsants, antidepressants, and analgesics. Fewer than ten reports of OC/drug interactions were found involving antihistamines, thyroid hormone, vitamin C, antacids, ulcer medication, or diuretics. These may represent a set of OC/drug interaction problems that need to brought into medical awareness. Pregnancy is the first event reported when OCs appear to interact with another drug. However, menstrual disturbances are reported more often. BTB is the most frequently reported menstrual disturbance: it has been considered a warning signal that OC efficacy may be compromised. In such circumstances, contraceptive backup may be warranted. Reports of interference with OC efficacy have been most common for drugs used to treat tuberculosis, epilepsy, and depression, so patients and their physicians should be aware of potential problems. However, the average woman is more likely to encounter antibiotics, analgesics, and antihistamines, and current package inserts contain appropriate warnings. In recent years, prescriptions for low-estrogen OCs have outnumbered those for high-dose preparations. Many physicians became concerned that there was an increased risk of OC drug failure with the low-dose products. The database does not seem to suggest that this has happened. The dose of estrogen is not correlated with total adverse experience reports, time of appearances of the first adverse experience reports, or rate of reporting of the interactions. Likewise, reports of potential interactions with menstrual disturbances are not correlated with lower estrogen doses in OCs. There is, however, an association between low-estrogen OCs and recently reported pregnancies attributed to OC/drug interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

PIP: Given the large numbers of oral contraceptive (OC) users, coupled with the prevalence of use of antibiotics, antidepressants, anticonvulsants, and anticoagulants, there is a need for a comprehensive analysis of potential OC/drug interactions. This review includes a compilation of a database of all the adverse experience reports contained in the literature, results voluntarily reported to Syntex, and a survey of clinical and animal studies in this field. Over 700 alleged OC/drug interactions were reported for antituberculous drugs, other antibiotics, anticonvulsants, antidepressants, and analgesics. Fewer than 10 reports of OC/drug interactions were found involving antihistamines, thyroid hormones, vitamin C, antacids, ulcer medication, or diuretics. The greatest number of incidents (256) involved antibiotics; of these, antituberculous drugs accounted for 76% of the total. Of the 713 cases involving possible interference with the efficacy of OCs, only 21% resulted in pregnancies; 41% reported menstrual disturbances and 38% reported no problems at all. 94% of the cases of adverse OC/drug interactions involved higher-dose (50 mcg or more) estrogen OCs. Likewise, reports of potential interactions with menstrual disturbances are not correlated with lower estrogen doses in OCs. There is, however, an association between low-estrogen OCs and recently reported pregnancies attributable to OC/drug interactions. Breakthrough bleeding was the most commonly reported menstrual disturbance and may constitute a warning sign of adverse OC/drug interactions.

Biosynthesis of the major urinary proteins in mouse liver: a biochemical genetic study.

By labeling liver protein in vivo with [3H] leucine, the relative biosynthetic rate has been measured for the major urinary proteins (MUPs), three closely related, androgen-regulated proteins that are synthesized in mouse liver, secreted into the bloodstream, and excreted into the urine. In livers from females of strain C57BL/6J, total MUP synthesis represents about 0.6-0.9% of the total protein synthesis; in males and testosterone-treated females of the same strain, synthesis increases to about 3.5-4.0% of the total. This 4- to 6-fold induction of total MUP synthesis is similar to the androgen-mediated increase in MUP-specific messenger RNA reported by others, and indicates that the previously observed 20- to 25-fold induction of total MUP excretion into urine is generated partly at the posttranslational level. By measuring the ratio of synthesis of the individual MUPs, it was determined that the testosterone-mediated change in the pattern of MUP synthesis, indicating nature, of MUPs excreted. A survey of seven inbred mouse strains revealed polymorphism for the rate of total MUP synthesis in untreated females. Two classes could be distinguished on the basis of a 3-to 5-fold difference in the rate. This variation does not correlate with variation at Mup-a, a locus that controls the ratio of the three MUPs in urine from androgen-induced mice. These findings are consistent with the notion that MUP expression is controlled by a variety of independently assorting genes.

A general method for retrieving the components of a genetically engineered fusion protein.

Escherichia coli expression vectors encoding an acid-labile aspartyl-proline (Asp-Pro) dipeptide bridging two protein sequences were constructed and used to synthesize two different bovine growth hormone (bGH) fusion proteins. The codons GAT-CCX coding for Asp-Pro are provided by the recognition site for Bam HI (GGATCC). Treatment of the bGH fusion proteins at low pH in the presence of guanidine hydrochloride releases the bGH moiety from the fusion protein. The release of the bGH from the fusion protein specifically requires the Asp-Pro dipeptide linking the bGH sequence to the fusion protein. The bGH released from the fusion protein retains anti-bGH immunoreactivity as well as the ability to bind to growth hormone receptor in vitro.

Human parathyroid hormone gene (PTH) is on short arm of chromosome 11.

The human gene for parathyroid hormone (PTH) was chromosomally mapped using human-rodent hybrids and Southern filter hybridization of cell hybrid DNA. A recombinant DNA probe containing human PTH cDNA insert (pPTHm122) hybridized to a 3.7-kb fragment in human DNA cleaved with the restriction enzyme EcoRI. By correlating the presence of this fragment in somatic cell hybrid DNA with the human chromosomal content of the hybrid cells, the PTH gene was mapped to the short arm of the chromosome 11.