Quantification of changes in c-myc mRNA levels in normal human bronchial epithelial (NHBE) and lung adenocarcinoma (A549) cells following chemical treatment.

Lung tumors frequently exhibit altered expression of oncogenes and/or tumor suppressor genes. Although some of these alterations are believed to arise from chemical exposure, the ability of specific chemicals to cause distinct changes in gene expression is not well characterized. We previously reported the development of a quantitative reverse transcriptase/polymerase chain reaction (RT/PCR) method for measuring c-myc mRNA levels, and reported that c-myc proto-oncogene expression is significantly increased in small-cell lung carcinoma cells. In the present study, quantitative RT/PCR was used to assess the effect of model toxins cycloheximide (CHX), a protein synthesis inhibitor, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a DNA alkylating agent, on c-myc mRNA levels in normal human bronchial epithelial (NHBE) and lung adenocarcinoma (A549) cells. Expression of c-myc was evaluated at 1-100 microM CHX and MNNG and was compared to the cytotoxic response as measured by the neutral red assay. Cycloheximide elicited a dose-dependent increase in c-myc mRNA levels in NHBE and A549 cells, but did not alter expression of the housekeeping gene beta-actin. A maximum increase for c-myc expression (200% of control) was observed 5 h after treatment with noncytotoxic concentrations. In contrast, MNNG elicited a dose-dependent decrease in c-myc expression in A549 cells, but no significant change in c-myc was observed in NHBE cells. The results from this study suggest that the quantitative RT/PCR method may be an appropriate technique for monitoring gene expression changes following chemical exposure. Hence, these types of studies may assist in the identification of specific chemicals which may induce the genetic alterations involved in the development of lung cancer as well as provide information relevant to the interactive effects of chemicals within complex mixtures.

Gene expression profiling of cultured human bronchial epithelial and lung carcinoma cells.

Lung cancer is a complex collection of diseases that is thought to begin with single mutated progenitor cells and culminates in any of several clinically described pathologies. Our knowledge of the molecular events that lead to different lung cancer types--small cell carcinoma, squamous cell carcinoma, adenocarcinoma, and large cell carcinoma--is incomplete. Nonetheless, it is evident that genetic changes that impact multiple molecular networks are involved in the generation of each specific phenotype. Due to the obvious complexity of these processes, the simultaneous quantitative monitoring of changes in the expression of genes that define these networks can provide mechanistic information to increase our understanding of the molecular basis for human pulmonary carcinogenesis. To this end, we have employed a commercially available human cDNA array (Atlas Human Array, Clontech Laboratories) to systematically screen for alterations in the expression of 600 genes in normal human bronchial epithelial (NHBE) cells as well as in several lung carcinoma lines. Studies on the reproducibility and variability of array results indicate that a 2-fold or greater difference in the expression of a particular gene could be considered a real difference in transcript abundance. Accuracy of gene expression as measured in the array was verified by comparing mRNA levels of the proto-oncogene c-myc in the array with results obtained by traditional Northern blot analysis and by quantitative RT-PCR. Gene expression profiles were compared within and among cell types. The differential expression of 17 genes, including downregulation of MRP8 and MRP14 and upregulation of CYP1B1, was observed in all four carcinoma lines compared to NHBE cells. The direction of all 17 gene expression differences, either upregulation or downregulation relative to NHBE cells, was the same for all four carcinoma lines, underscoring their common molecular features. Each lung tumor line also exhibited a number of unique differences compared to both normal cells and the other tumor cell lines. These differences may be due to differences in the cellular origin and/or pathology of the cell lines studied.

Expression of stably transfected murine glutathione S-transferase A3-3 protects against nucleic acid alkylation and cytotoxicity by aflatoxin B1 in hamster V79 cells expressing rat cytochrome P450-2B1.

Aflatoxin B1 (AFB1) is activated to AFB1-8,9-oxide (AFBO), a potent mutagenic and carcinogenic metabolite of AFB1. In the mouse, AFBO has been shown to be most efficiently detoxified by a specific isozyme of alpha-class glutathione S-transferase (GST), mGSTA3-3 (mGST-Yc). A hamster V79 cell line (V79MZr2B1, originally designated V79/SD1) previously transfected with the rat cytochrome P450-2B1 was stably transfected with an mGSTA3-3 expression vector, to study the chemopreventive role of GST in protecting against cytotoxicity or genotoxicity of AFBO. Immunoblotting demonstrated strong expression of an alpha-class GST in the mGSTA3-3 transfected cell line, whereas no detectable alpha-class GST protein was observed in the control (empty vector-transfected) cells. Previous studies with the V79MZr2B1 cell line indicated that it can activate AFB1 to a mutagenic metabolite via a transfected rat P450-2B1 stably expressed in the cells. We examined the ability of the expressed mGSTA3-3 to protect against AFB1-induced cytotoxicity or [3H]-covalent adduct formation in cellular nucleic acids. Exposure of empty vector-transfected control cells and mGSTA3-3 expressing cells to up to 600 nM [3H]-AFB1 indicated that a 70-80% reduction in DNA and RNA adducts was afforded by the expression of mGSTA3-3 in the transfected cells. Clonogenic survival assays showed that the mGSTA3-3 cell line was 4.6-fold resistant to AFB1 cytotoxicity as compared with the empty vector-transfected control SD1 cells, with IC50 values of 69 and 15 microM, respectively. The results of these studies demonstrate that mGSTA3-3 confers substantial protection against nucleic acid covalent modification and cytotoxicity by AFB1 in this transgenic cell model system.

Overexpression of stably transfected human glutathione S-transferase P1-1 protects against DNA damage by benzo[a]pyrene diol-epoxide in human T47D cells.

The (+)-anti enantiomer of benzo[a]pyrene-7,8-dihydrodiol-9, 10-epoxide (BPDE) is a potent mutagenic and carcinogenic metabolite of benzo[a]pyrene (BP), and a major fraction is conjugated with glutathione in vivo. The chemopreventive role of glutathione S-transferases (GSTs) in protecting against covalent modification of DNA and other cellular macromolecules by BPDE was modeled in human T47D and MCF-7 cell lines previously stably transfected with human GSTpi1 (hGSTP1). Cells were exposed to [3H]BPDE (30-600 nM). Dose-response experiments indicated that the high level of expression of hGSTP1-1 in the T47Dpi cell line (4411 +/- 183 milliunits/mg of cytosolic protein, using 1-Cl-2,4-dinitrobenzene as substrate), resulted in 70-90% reduction in the covalent 3H-adduct formation in DNA or RNA isolated from the GSTP1-transfected T47Dpi cell line. The lower level of hGSTP1-1 expression in the transfected MCF-7 cell line (91 milliunits/mg) provided only marginal protection against [3H]BPDE adduct formation and did not affect sensitivity to BPDE-induced cytotoxicity. Protection against BPDE-induced cytotoxicity was observed only in the T47Dpi cell line, which had an IC50 value 5.8-fold greater than that of the T47Dneo control cell line. Measurement of glutathione conjugates of BPDE indicated that the total conjugation was 5-fold higher in the GSTpi-transfected T47D line, most of which was exported into the culture medium over the 20-min exposure period. These results indicate that hGSTP1-1 protects effectively against DNA and RNA modification by BPDE, but moderate to high level expression may be required for strong protection against BPDE-induced genotoxicity and cytotoxicity.

Chemoprotective functions of glutathione S-transferases in cell lines induced to express specific isozymes by stable transfection.

The authors have shown that expression of mGSTM1-1 or hGSTP1-1 in MCF-7 cells protects against DNA alkylation by 4-nitroquinoline-1-oxide (NQO) in an isozyme-specific manner and is commensurate with relative specific activity. Expression of GSTs also conferred protection against both DNA strand breaks and sister-chromatid exchange induced by NQO. Interestingly, GST expression did not protect against NQO cytotoxicity in transfected MCF-7 cell lines, although resistance to NQO cytotoxicity was observed in a T47D pi transfectant line, expressing much higher specific activity of the transfected hGSTP1-1. However, high level expression of hGSTP1-1 or mGSTM1-1 in V79 transfectants did not confer resistance to cytotoxicity, indicating that expression of GST alone is not sufficient. The authors have also shown protection against AFB1 in cell lines expressing transfected rat CYP2B1 (V79MZr2B1) and transfected mGST-Yc (mGSTA3-3). Protection was observed against both alkylation of DNA (3-fold) by [3H]AFB1 and against AFB1 cytotoxicity (7-fold). Similarly, V79MZr1A1 cells that express CYP1A1 and either transfected human or murine GSTP1-1 (< 5000 mIU/mg, CDNB) exhibited > 70% decrease in covalent labeling of total nucleic acids by [3H]BPDE. However, no protection against the cytotoxicity of BPDE was conferred by expression of hGSTP1-1. Overall, these results indicate that in some (NQO or BPDE), but not all (AFB1) cases, protection by GST expression against DNA damage is more effective than protection against cytotoxicity. In addition, there is evidence to indicate that additional factor(s) other than high GST isozyme expression level and good substrate efficacy affect the degree of protection against cytotoxicity of reactive electrophiles. This includes the differential protection against NQO cytotoxicity in T47D pi, but not V79 Xh pi-33 cells and also the recent studies which showed that expression of the MRP GS-X conjugate efflux transporter confers synergistic protection against NQO cytotoxicity when co-expressed with transfected human GSTP1-1 in MCF-7 cells. Thus, protective efficacy conferred by GST expression can vary with different cellular targets and/or experimental end-points, as well as with variations in relative specific activity or in different cellular phenotypic contexts.

Protection by transfected glutathione S-transferase isozymes against carcinogen-induced alkylation of cellular macromolecules in human MCF-7 cells.

Increased expression of glutathione S-transferase (GST) isozymes has been correlated with development of resistance both to cytotoxic anticancer agents and to genotoxic carcinogens. While most anticancer agents are poor GST substrates, the model alkylating carcinogen 4-nitroquinoline-1-oxide (NQO) is a good substrate for human pi class GST (hGSTP1-1) and murine GST mu-1 (mGSTM1-1), but not human GST alpha-2 (hGSTA2-2). We investigated whether expression of these GST isozymes in stably transfected clonal cell lines could protect against the genotoxic and cytotoxic effects of NQO. Compared to parental MCF-7 or pSV2neotransfected control cell lines, covalent labeling of total cellular macromolecules by [3H]NQO (0.1-1.0 mM) was reduced by 70% and 92% in hGSTP1-1- and mGSTM1-1-transfected cell lines, respectively, but was not affected in the hGSTA2-2 expressing line. The observed protection was closely correlated with the relative specific activity of each cell line for conjugation of NQO by the transfected GST isozymes and this protection was reversible by pretreatment of cells with the GST inhibitor ethacrynic acid. Similar results were obtained when covalent labeling of total cellular nucleic acid or DNA was measured. However, clonogenic survival assays indicated that the sensitivity of these cell lines to the cytotoxic effects of NQO was similar for the control and GST-transfected MCF-7 cell lines. Thus, while expression of hGSTP1-1 and mGSTM1-1 (but not hGSTA2-2) was highly protective against alkylation of cellular macromolecules by NQO, this protection was not effective against cytotoxicity induced by NQO as measured by clonogenic assay. These results indicate that expression of GST isozymes can protect differentially against the acute genotoxic and potentially mutagenic effects, as compared to the cytotoxic effects, of electrophiles that are detoxified by glutathione conjugation.

Inflammatory aneurysm of the abdominal aorta: diagnosis by computerized tomography and ultrasonography.

We correctly diagnosed seven cases of inflammatory aneurysm of the abdominal aorta preoperatively by computerized tomography (CT) and ultrasonography (US). Excessive thickening of the aneurysmal wall and conspicuous enhancement on CT are the characteristic features that led to the correct diagnosis. Ultrasonographic findings are nonspecific, but US is the screening method of choice. If US shows a sonolucent zone anterior or anterolateral to an atherosclerotic aneurysm, CT should be used to delineate the perivascular abnormalities.

Real-time renal sonography in spinal cord injury patients: prospective comparison with excretory urography.

A prospective blinded comparison of real-time renal sonography and excretory urography was done in 202 urologically asymptomatic patients with spinal cord injury who underwent periodic evaluation. Sonography identified 31 of 31 renal masses (100 per cent) (30 cysts and 1 xanthogranulomatous pyelonephritis), whereas excretory urography identified 14 of 31 masses (45 per cent). Of the 398 kidneys evaluated hydronephrosis owing to an obstructive etiology was noted in 7, all of which (100 per cent) were identified on excretory urography and 6 (86 per cent) were identified on sonography. Only 12 of the 48 kidneys (25 per cent) with typical changes of chronic pyelonephritis on excretory urography were diagnosed correctly by ultrasound. Sonography identified 18 of 23 kidneys (78 per cent) with calculi compared to 20 of 23 (87 per cent) by excretory urography. Although 127 abnormalities were noted in 202 patients, only 21 dictated a change in management. Thirteen abnormalities were visible on a plain film of the kidneys, ureters and bladder (3 kidneys with stones, 1 ureteral stone and 9 bladders with stones). We conclude that sonography and excretory urography are excellent diagnostic modalities for the evaluation of the kidneys. Sonography, plain radiograph of the abdomen and post-contrast injection x-rays on excretory urography frequently offer complementary diagnostic information. The noninvasive nature of ultrasound examination and lack of x-ray exposure combined with no need for special patient preparation make ultrasound examination extremely attractive in this patient population. It is recommended that real-time renal ultrasound and a plain radiograph of the abdomen be used on an alternate basis with excretory urography for the routine followup of spinal cord injury patients.

Low-dose fibrinolytic therapy in hand ischemia.

Intra-arterial, low-dose streptokinase therapy was administered to five patients with acute hand ischemia (2 to 48 hours' duration) and one patient with chronic hand ischemia (3 months' duration). In the five patients with acute ischemia excellent restoration of patency of occluded hand arteries was obtained, with complete recovery of hand function and avoidance of tissue loss despite the severity of these cases. In the remaining patient with an iatrogenic chronic occlusion, ischemia could not be relieved and bypass surgery was performed with satisfactory results noted at the 3-month follow-up examination. No complications were encountered during or after any of these procedures. The catastrophic consequences of hand ischemia, i.e., loss of limbs or digits, were avoided.

Noninvasive assessment of the transplanted kidney: ultrasound vs. computerized tomography.

In our experience both ultrasound and CT are highly diagnostic in the identification of the posttransplant fluid collections and hydronephrosis. As is the case elsewhere in the abdomen, the two examinations appear to be complementary. Ultrasound should be the initial diagnostic modality due to the advantages mentioned before; CT is helpful in resolving questionable abnormalities noted on ultrasound or in difficult clinical situations. Combination of the two examinations in selected cases improves sensitivity and specificity in the detection of postoperative complications.

Acquired double pylorus due to gastroduodenal fistula complicating peptic ulceration.

Fistulous communication between the gastric antrum and the duodenal bulb had the roentgenographic appearance of a double-channel pylorus in five adult patents. A penetrating peptic ulcer was the underlying cause in each instance. Following conservative management, the fistula in two cases closed spontaneously and in two other cases became asymptomatic despite persistence of the accessory channel. The remaining patient underwent subtotal gastrectomy because of hemorrhage from nonhealing antral ulcer.

Evaluation of retroperitoneal hemorrhage by computed tomography before and after translumbar aortography.

Twenty patients were prospectively studied by computed tomography (CT) before and after undergoing translumbar aortography (TLA). Changes indicative of retroperitoneal bleeding were depicted by CT in all 20 patients despite the predominantly small size of the hematomas. CT scans obtained within two hours after TLA demonstrated: (a) thickening of the diaphragmatic crura, (b) enlargement of the left psoas muscle, and (c) obscuration of the aortic outline by soft-tissue density. Follow-up scans at 24 hours (10 patients) and one week (3 patients) revealed marked decrease in abnormalities, suggesting rapid resorption of the hematoma.

Tuberculous mediastinal lymphadenopathy in adults.

Three cases of paratracheal and hilar lymph node enlargement without parenchymal lung lesion simulating sarcoidosis, lymphoma, and metastasis were finally proved to be tuberculous lymphadenitis. Although it is not a common manifestation of tuberculous infection in the adult, this possibility should be considered in the differential diagnosis of mediastinal lymphadenopathy, especially in non-whites.