An investigation into the suitability of a commercial real-time PCR assay to screen for Taylorella equigenitalis in routine prebreeding equine genital swabs.

REASONS FOR PERFORMING STUDY: Standard bacteriological methods for identifying Taylorella equigenitalis in cervical smears are time consuming. Therefore, a more rapid real-time PCR assay was evaluated for its suitability in screening swabs. OBJECTIVE: To compare the results of a commercially available real-time PCR assay with routine microbiological culture for the identification of T. equigenitalis, the causative organism of contagious equine metritis, in equine genital swab samples, under 'field trial' conditions. MATERIALS AND METHODS: Routine prebreeding genital swabs (n=2072) collected from Thoroughbred mares and stallions during 2009 were examined together with stored T. equigenitalis positive material. Swabs were cultured for T. equigenitalis using standard microbiological techniques. Bacterial lysates were isolated from the swabs and examined for the presence of a 16S DNA fragment of T. equigenitalis, using a commercial multiplex real-time PCR assay system. RESULTS: There was complete concordance between positive and negative results obtained by the 2 methods. Real-time PCR also detected T. equigenitalis DNA from swabs that were negative using standard microbiological culture after 6 months' storage at +4 degrees C but from which T. equigenitalis had been isolated following collection. The sensitivities of real-time PCR and bacterial culture were both 10(-3) (equivalent to 3 colony-forming units). CONCLUSION AND CLINICAL RELEVANCE: Routine bacterial culture of T. equigenitalis requires an incubation period of not less than 7 days before a conclusive negative result can be obtained, whereas bacterial extraction and real-time PCR assay can be completed in less than 6 h. The commercially-available PCR assay tested provided a rapid and reliable method for the identification of T. equigenitalis from equine genital swabs and could be usefully employed for the screening of mares and stallions for preseason Horserace Betting Levy Board (HBLB) Code of Practice and in other situations such as for bloodstock sales screening requirements, overcoming the current delays imposed by bacterial culture requirements. Its use could be quality assured by the existing HBLB biannual testing scheme for designated laboratories.

Glutathione S-transferase: a potential new marker of intestinal ischemia.

BACKGROUND/PURPOSE: The accurate and early diagnosis of intestinal ischemia remains difficult chiefly because of a lack of a suitable marker that is noninvasive and easy to use. The glutathione S-transferases (GST) are a family of cytosolic enzymes involved in detoxification and released from a variety of cells when the cell membrane is damaged. The enzymes are distributed widely in the intestine and show isoform specificity in their distribution throughout the intestinal tract. Several previous reports have shown the utility of these enzymes in the diagnosis of liver and renal graft damage during and after organ transplantation. The object of this study was to determine if GST levels correlated with histologic changes of intestinal ischemia in a controlled animal model of mesenteric intestinal ischemia. METHODS: Control and experimental male Sprague-Dawley rats underwent laparotomy and ligation of the Superior Mesenteric Artery (SMA) and both control and experimental animals were studied at 30, 60, 90, 120, and 240 minutes. Blood taken from the Inferior Vena Cava (IVC) and Portal Vein (PV) and jejunal and ileal perfusates were assayed for alpha and mu isoforms of GST using a commercially available enzyme immunoassay. In addition, jejunal and ileal segments were sampled and reviewed by a histopathologist blinded to the group being studied. RESULTS: A reproducible pattern of intestinal ischemia was noted with worsening grades of injury observed with greater ligation times. Luminal alpha and mu GST release (as measured by the appearance in luminal perfusate) increased with increasing ischemia times. Increased ischemia times resulted in increased levels of alpha and mu GST in both portal and systemic venous samples but lagged behind the appearance of raised luminal GST values. CONCLUSIONS: The results suggest that GST may be an interesting and useful marker in the early detection of intestinal ischemia. Its detection in peripheral blood has implications for a more detailed study design to determine the sensitivity and specificity of this marker in more diverse clinical conditions such as necrotizing enterocolitis and superior mesenteric artery occlusion.

Differential regulation of calcitonin secretion in normal and neoplastic pulmonary neuroendocrine cells in vitro.

Within the mammalian lung, cells with a neuroendocrine phenotype are few in number and are sparsely distributed. In contrast, neuroendocrine neoplasms represent a major group of lung cancers. The aim of this study was to develop a model of mammalian PNECs and to compare glucocorticoid regulation of calcitonin secretion in normal and neoplastic cells with neuroendocrine differentiation. Cell cultures of PNECs were initiated after the disaggregation of neonatal hamster lungs with 0.1% collagenase and fractionation of the resultant cell suspension on a gradient of iodixanol (1.320 g/mL). Cell fractions enriched in PNECs were identified by positive staining for 5-hydroxytryptamine and the presence of calcitonin. Calcitonin secretion was investigated after exposure to hydrocortisone (0 to 1,000 nM). A dose-dependant inhibition of calcitonin secretion was seen after 7 days between 10 nM (55% of control), and 1,000 nM (29%) hydrocortisone. Cell cultures grown in the presence of hydrocortisone also contained significantly fewer PNECs between 10 nM (90% of control), and 1,000 nM (45%). Human bronchial carcinoid cells (NCIH727) cultured under identical conditions showed a similar inhibition of calcitonin secretion between 10 nM (53%) and 1,000 nM (52%), although at these concentrations, no reduction in cell number was seen. In contrast, 2 human small cell lung cancer cell lines (DMS-79 and COR-L24 cells) showed no dose-dependent inhibition of calcitonin secretion and no effect on cell proliferation in response to hydrocortisone. These results show that enriched cultures of mammalian PNECs can be used to investigate functional aspects of their biology, including peptide secretion in response to potential regulators. Furthermore, calcitonin secretion is inhibited in normal PNECs and bronchial carcinoid cells at physiological concentrations of glucocorticoids, but this feature appears not to be present in the 2 more invasive neuroendocrine neoplasms (small cell lung cancer cells) investigated in this study.

Localized conjunctival argyrosis: a late sequela of strabismus surgery.

We describe a case of focal argyrosis of the conjunctiva clinically simulating a melanoma. An 82-year-old woman was referred for an asymptomatic pigmented conjunctival lesion. Her only significant past ocular history was strabismus surgery 76 years earlier. Biopsy of the conjunctiva and lateral rectus muscle revealed the discoloration was pigment granules. Energy-dispersive x-ray microanalysis revealed the pigmentation to be silver deposits. The patient had strabismus surgery probably using a silver clip. Argyrosis should be considered in the differential diagnosis of focal pigmented conjunctival lesions.

DNA repair: kinetics and thresholds.

DNA damage is a critical factor in the initiation of chemically induced toxicities (including cancer), and the repair of this damage represents the cell's first line of defense against the deleterious effects of these agents. The various mechanisms of DNA repair are reviewed briefly and the actions of the DNA repair protein O6-alkylguanine DNA alkyltransferase (ATase) are used to illustrate how DNA repair can protect cells against alkylating agent-induced toxicities, mutagenesis, clastogenesis, and carcinogenesis. The effectiveness of this repair protein can be measured based on its ability to deplete levels of its promutagenic substrate O6-methylguanine (O6-meG) in the DNA of cells. These studies reveal that the repair of O6-meG from DNA occurs heterogeneously, both intra- and intercellularly. Even in cells that repair O6-meG hyperefficiently, certain regions of chromatin DNA are repaired with difficulty, and in other regions they are not repaired at all; most likely this lack of repair is a result of the location of the lesion in the DNA sequence. When individual cells are compared within a tissue, some cells are clearly repair deficient, because the O6-meG can persist in DNA for many weeks, whereas in other cells, it is removed within a matter of hours. The role of these repair-deficient cells as targets for alkylating agent induced carcinogenesis is considered. The mechanisms of the homeostatic control of DNA repair function in mammalian cells are not yet well understood. Because there are now indications of the mechanisms by which the level of DNA damage may be sensed (and so influence the activity of the ATase repair protein), this is an important area for future study.

Overexpression of the glucose-regulated stress gene GRP78 in malignant but not benign human breast lesions.

The 78 kDa glucose-regulated stress protein GRP78 is induced by physiological stress conditions such as hypoxia, low pH, and glucose deprivation which often exist in the microenvironments of solid tumors. Activation of this stress pathway occurs in response to several pro-apoptotic stimuli. In vitro studies have demonstrated a correlation between induced expression of GRP78 and resistance to apoptotic death induced by topoisomerase II-directed drugs. We were interested in characterizing this protein in human breast lesions for potential implications in chemotherapeutic intervention. Surgical specimens of human breast lesions and paired normal tissues from the same patients were flash frozen for these studies. Total RNA and/or protein were extracted from these tissues and used in northern and/or western blot analyses, respectively, to quantify the relative expression of GRP78. Northern blot analysis indicated that 0/5 benign breast lesions, 3/5 estrogen receptor positive (ER+) breast tumors, and 6/9 estrogen receptor negative (ER-) breast tumors exhibited overexpression of GRP78 mRNA compared to paired normal tissues, with fold overexpressions ranging from 1.8 to 20. Western blot analyses correlated with these findings since 0/5 benign breast lesions, 4/6 ER+ breast tumors, and 3/3 ER- breast tumors overexpressed GRP78 protein with fold overexpressions ranging from 1.8 to 19. Immunohistochemical analysis of these tissues demonstrated that the expression of GRP78 was heterogeneous among the cells comprising different normal and malignant glands, but confirmed the overexpression of GRP78 in most of the more aggressive ER- tumors. These results suggest that some breast tumors exhibit adverse microenvironment conditions that induce the overexpression of specific stress genes that may play a role in resistance to apoptosis and decreased chemotherapeutic efficacy.

Plasma concentrations of glutathione S-transferase isoenzyme are raised in patients with intestinal ischaemia.

BACKGROUND: The mortality rate associated with acute mesenteric ischaemia (AMI) remains high. Diagnosis is frequently confounded by the non-specific history and physical signs, in conjunction with the absence of a reliable biological assay. Glutathione S-transferase (GST) is an enzyme with a crucial role in cellular homoeostasis, the alpha isoenzyme of which is highly specific to small bowel and liver. This study assessed alphaGST as a marker for AMI. METHODS: Twenty-six patients with acute abdominal pain were enrolled. Each patient manifested a diagnostic dilemma, with a potential diagnosis of AMI. Plasma was reserved for alphaGST assay during routine blood testing and stored at -20 degrees C for analysis. A final diagnosis was made by autopsy, laparotomy, a definitive other investigation or a return to full health. RESULTS: Twelve patients had AMI. Plasma alphaGST was significantly increased in patients with AMI (P < 0.0001). Although pH differed and other biochemical changes occurred, only alphaGST accurately predicted AMI. CONCLUSION: A threshold of 4 ng/ml for alphaGST was 100 per cent sensitive and 86 per cent specific for AMI. If these observations can be confirmed, evaluation of alphaGST may reliably predict the presence or absence of AMI.

Arrest of replication by mammalian DNA polymerases alpha and beta caused by chromium-DNA lesions.

We have previously shown that trivalent chromium, and hexavalent chromium in the presence of one of its primary in vivo reductants, ascorbate, can bind to DNA and form interstrand crosslinks capable of obstructing replication. This effect was demonstrated in vitro by using Sequenase Version 2.0 T7 DNA polymerase; its parent enzyme, the unmodified T7 DNA polymerase; and Escherichia coli polymerase I large (Klenow) fragment; and it was demonstrated ex vivo by using Taq polymerase and DNA from chromium-treated human lung cells as template. This study was performed to determine whether DNA-bound chromium affects mammalian DNA polymerases in the same manner. Two mammalian enzymes, DNA polymerase alpha and DNA polymerase beta, were used. DNA polymerase alpha is a processive enzyme believed to be the primary lagging-stand synthetase, whereas DNA polymerase beta is a non-processive enzyme believed to function in DNA repair by filling single stranded gaps one base at a time. DNA polymerase arrest assays were performed with each of these enzymes to replicate DNA with toxicologically relevant levels of chromium adducts produced by either trivalent chromium or hexavalent chromium and ascorbate. Both enzymes responded to chromium-DNA damage by arresting replication, and the arrests increased in a dose-dependent manner. Furthermore, the guanine-specific pattern of arrests produced when an exonuclease-free preparation of DNA polymerase beta was used corresponded exactly to the arrest patterns produced in vitro by the exonuclease-free enzyme Sequenase and ex vivo by Taq polymerase. These results suggest that replication arrest may be a common response of polymerases to DNA-chromium lesions and provide a plausible mechanism for the inhibition of DNA synthesis and S-phase cell-cycle delay that occurs in mammalian cells treated with genotoxic chromium compounds.

Glutathione S-transferases as biomarkers of organ damage: applications of rodent and canine GST enzyme immunoassays.

The cytosolic glutathione S-transferase (GST) enzymes serve as ideal biomarkers of organ damage as they exhibit many of the required characteristics, i.e. specific localisation, high cytosolic concentration and relatively short half-life. The role of GSTs as early indicators of organ damage is applicable to both human and animal models. Because of the regio-specific localisation of the different isoforms of GST in liver and kidney, simultaneous monitoring of classes of GSTs in biological matrices permits the identification of specific areas of damage within a particular organ. Immunoassays have been developed which quantify canine alpha GST and roden microGST (Yb1). The immunoassays are solid phase EIAs, where GST in the sample or standard is captured by a specific anti-GST antibody coated onto the solid phase. After washing, a specific enzyme-labelled IgG conjugate is added which binds to the captured GST. After a further washing step, substrate is added and a colour developed. The absorbance is measured on an ELISA plate reader and is directly proportional to the amount of GST present in the sample. The assays are performed at room temperature and can be completed within 3 h. The immunoassays are specific for each GST and have a range of 0-100 micrograms/l. A range of assay parameters were investigated to validate the EIAs for GST detection. The assays are sensitive and reproducible. CV for inter- and intra-assay variation were below 9% for Yb1 assay and below 20% for the canine alpha GST EIA. Recovery of spiked GST over the standard curve range was 102 and 99%, respectively. No prozone effect was observed and samples exhibited linearity of dilution in both assays. Validation has shown that using these enzyme immunoassay, Yb1 and canine alpha GST can be measured accurately and precisely in biological matrices, tissue homogenates and cell lines and that changes in GST levels can be detected. The use of these assays have important applications in both in vitro and in vivo toxicity studies, where GST's serve as sensitive marker of hepatocellular and renal cell integrity.

Atraumatic fetal cervical spinal cord injury and cruciate paralysis.

BACKGROUND: Congenital cervical spinal cord injury usually is attributed to intrapartum mechanical trauma. CASE: A fetal cervical spinal cord hemorrhage presented as congenital isolated upper extremity ("cruciate") paralysis and muscle atrophy after an atraumatic delivery. Timely neonatal diagnosis and surgery resulted in complete recovery. CONCLUSION: Paralysis may result from atraumatic spinal cord injuries occurring in the antepartum period.

Apoptosis: inhibitor or instigator of carcinogenesis?

Carcinogenesis is considered to require an initiating event that results in an irreversible genetic change in a subpopulation of cells. Based on the available evidence, it seems likely that apoptosis may act to attenuate this process by causing the deletion of genetically damaged cells from the host organism. Nevertheless, the existence of an active pathway leading to apoptotic cell death may be a double-edged sword, simply because it can be overcome. Some cells may exhibit preexisting genetic or epigenetic insensitivity to induction of apoptosis. Surviving cells may contain sub- lethal levels of DNA damage and be induced to proliferate as an indirect result of the carcinogen-induced apoptotic cell death of surrounding tissue. This process would facilitate the acquisition mutations in the genome, possibly resulting in further insensitivity to apoptosis through activation of the bcl-2 oncogene or inactivation of the p53 tumor suppressor gene. In this context, the propensity of a cell to undergo apoptosis could be viewed as a selection pressure that a tumor cell must overcome. For neoplastic growth to occur, an imbalance between proliferation and apoptosis must be established such that cell growth predominates. Genetic mutations or epigenetic factors that diminish the propensity of a cell to undergo apoptosis may therefore confer on that cell a growth advantage.

Cytomegalovirus keratitis after penetrating keratoplasty.

We report the development of cytomegalovirus (CMV) keratitis in the penetrating keratoplasty of a 59-year-old human immunodeficiency virus-negative woman after uncomplicated corneal transplantation. Immunosuppression with topical cyclosporine A 2% in corn oil and topical prednisolone acetate 1% suspension was used postoperatively. The 15-month postoperative course was complicated by multiple episodes of endothelial rejection, medically controlled elevated intraocular pressure, polymicrobial bacterial (coagulase-negative staphlococcus and alpha-hemolytic streptococcus) keratitis, and endothelial plaque formation with associated hypopyon and epithelial defect. The graft failed and penetrating keratoplasty was repeated. Cytomegalovirus infection of superficial keratocytes in a region of scarring was identified in histological sections stained with hematoxylin and eosin and confirmed using mouse monoclonal anti-cytomegalovirus antibodies. Excision of the diseased corneal button with no additional treatment appears to have been curative. Low-grade keratitis was the only manifestation of the CMV infection, and it has not recurred 6 months postoperatively.

Base-specific arrest of in vitro DNA replication by carcinogenic chromium: relationship to DNA interstrand crosslinking.

We have previously shown that trivalent chromium can bind to purified DNA and form lesions capable of obstructing DNA replication in vitro. Trivalent chromium is not, however, carcinogenic to humans. Rather, it is the end product of the intracellular reduction of hexavalent chromium, which is carcinogenic. The process of chromium reduction yields several reactive intermediates which may also interact with DNA, perhaps producing different lesions than those generated when trivalent chromium binds DNA. The present study was undertaken to determine whether the treatment of DNA with hexavalent chromium in the presence of ascorbate (the intracellular reductant responsible for most in vivo chromium reduction), would also generate DNA lesions capable of obstructing replication. Using increasing chromium concentrations and a constant ascorbate:chromium ratio of 0.5:1 to generate biologically relevant adduct levels, a DNA polymerase arrest assay revealed that polymerase arresting lesions were formed and were indistinguishable from those generated by trivalent chromium, in that the most prominent arrests sites were one base upstream of guanine residues on the template strand. Measurement of the amount of chromium bound to template DNA in relation to the number of arrests demonstrated that only a subset (18.5%) of the chromium adducts were capable of causing polymerase arrest. Arrest assays performed with increasing ratios of ascorbate to chromium showed that high ratios (> or = 5:1) resulted in decreased polymerase arrests. DNA interstrand crosslinks in the arrest assay template were detected by renaturing agarose gel electrophoresis, and were shown to decrease markedly with increasing ascorbate to chromium ratios, whereas chromium binding levels remained unchanged. These results strongly implicate DNA interstrand crosslinks as the polymerase arresting lesion. The present study confirms and extends our previous study with trivalent chromium, and suggests that while the initial chemical nature of the DNA lesions formed by either trivalent chromium or reductive intermediates of hexavalent chromium may differ, their effect on DNA replication is the same.

Induction of internucleosomal DNA fragmentation by carcinogenic chromate: relationship to DNA damage, genotoxicity, and inhibition of macromolecular synthesis.

Hexavalent chromium (Cr) compounds are respiratory carcinogens in humans and animals. Treatment of Chinese hamster ovary cells with 150 and 300 microM sodium chromate (Na2CrO4) for 2 hr decreased colony-forming efficiency by 46 and 92%, respectively. These treatments induced dose-dependent internucleosomal fragmentation of cellular DNA beyond 24 hr after chromate treatment. This fragmentation pattern is characteristic of apoptosis as a mechanism of cell death. These treatments also induced an immediate inhibition of macromolecular synthesis and delayed progression of cells through S-phase of the cell cycle. Cell growth (as evidenced by DNA synthesis) was inhibited for at least 4 days and transcription remained suppressed for at least 32 hr. Many of the cells that did progress to metaphase exhibited chromosome damage. Chromate caused the dose-dependent formation of DNA single-strand breaks and DNA-protein cross-links, but these were repaired 8 and 24 hr after removal of the treatment, respectively. In contrast, Cr-DNA adducts (up to 1/100 base-pairs) were extremely resistant to repair and were still detectable even 5 days after treatment. Compared with other regions of the genome, DNA-protein cross-links and Cr adducts were preferentially associated with the nuclear matrix DNA of treated cells, which was 4.5-fold enriched in actively transcribed genes. Chromium adducts, formed on DNA in vitro at a similar level to that detected in nuclear matrix DNA, arrested the progression of a DNA polymerase in a sequence-specific manner, possibly through the formation of DNA-DNA cross-links.(ABSTRACT TRUNCATED AT 250 WORDS)

Preferential formation and repair of chromium-induced DNA adducts and DNA--protein crosslinks in nuclear matrix DNA.

The distributions of chromium-DNA adducts and DNA-protein crosslinks induced by treatment of intact CHO cells with carcinogenic chromium were examined in distinct chromatin subfractions: a chromatin subfraction released by digestion of isolated nuclei with micrococcal nuclease (1SF, 14% of total nuclear DNA), bulk chromatin (74% of total DNA) and a nuclear matrix fraction (12% of total DNA). The identity of the matrix fraction was confirmed by hybridization of DNA from each subfraction with a cDNA probe prepared from total mRNA isolated from CHO cells, which showed that the 1SF and nuclear matrix fractions were 2.3- and 3.8-fold enriched in actively transcribed genes respectively, compared to total unfractionated DNA. Immediately following treatment of cells with 150 microM sodium chromate for 2 h the binding of chromium to each chromatin fraction was found to be non-uniform. Compared with total unfractionated nuclei, the nuclear matrix fractions were enriched in chromatin-bound chromium (3.4-fold), whereas the bulk chromatin fraction was relatively depleted (0.5-fold). Approximately 13% of nuclear chromium was associated with the detergent-soluble lipid component of nuclei. A similar distribution of chromatin-bound chromium was also apparent 24 h after the chromate treatment. Immediately after the 2 h chromate treatment, chromium-DNA adducts were detected in all the chromatin subfractions. Total nuclear and bulk chromatin DNA contained similar levels of this type of damage. The 1SF fraction was depleted approximately 3-fold in this type of damage compared with total nuclear DNA. In contrast, the nuclear matrix was markedly enriched in chromium-DNA adducts (approximately 4-fold compared with total nuclear DNA) at this time. As previously demonstrated, chromium-DNA adducts in total nuclear DNA decreased within the first 24 h, but thereafter persisted at a similar level. Chromium-DNA adducts in nuclear matrix DNA also reached maximum levels at the end of the 2 h treatment and decreased to 68% and 39% of this level by 24 and 48 h after treatment respectively. In contrast, the adduct levels in the 1SF and bulk chromatin fractions did not change up to 48 h after treatment. Chromium-induced DNA-protein crosslinks, which were stable to 8 M urea and 2% SDS, occurred almost exclusively in the nuclear matrix fraction. The crosslinks in this fraction reached a maximum level at the end of the 2 h treatment, but returned to control levels 24 h later.(ABSTRACT TRUNCATED AT 400 WORDS)

Apoptosis is the mode of cell death caused by carcinogenic chromium.

The role of apoptosis in the mechanism of toxicity of hexavalent chromium, a human carcinogen, was investigated. Chinese hamster ovary (CHO) cells were treated with 150 or 300 microM sodium chromate for 2 hr, doses which decreased colony-forming efficiency to 53 and 5% of control, respectively. Cell growth was inhibited at least up to Day 8 after treatment. DNA synthesis was inhibited to 30 and 19% of control at 1 hr after treatment, and did not begin to recover until Day 4 after treatment. Protein synthesis was inhibited by 52 and 60% in 150 and 300 microM treated cells, respectively, 1 h after treatment, and recovered to 142 and 93%, respectively, at 24 hr. Incubation of cells with nontoxic doses of cycloheximide for 24 hr after treatment produced synergistic toxicity with chromate in colony-forming efficiency assays. Ion gradients persisted to Day 2 as revealed by exclusion of trypan blue dye in 97% of treated cells. Fluorescence microscopy of acridine orange-stained cells revealed morphological features of apoptosis including nuclear fragmentation in more than 90% of detached nonadherent cells and up to 22% of adherent cells by Day 2 after treatment. Untreated cells remained morphologically normal. Transmission electron microscopy of chromate treated cells showed characteristic features of apoptosis including chromatin margination and fragmentation, and cytoplasmic condensation with intact membrane and organelle structure. Internucleosomal DNA fragmentation (IDF) was delayed for at least 24 hr, whereafter it was detected in both adherent and nonadherent cells through Day 5 after treatment. These results indicate apoptosis as the mode of cell death caused by chromium and imply that apoptosis must be considered as a component of chromium-induced multistage carcinogenesis.

DNA polymerase arrest by adducted trivalent chromium.

Carcinogenic chromium (Cr6+) enters cells via the sulfate transport system and undergoes intracellular reduction to trivalent chromium, which strongly adducts to DNA. In this study, the effect of adducted trivalent chromium on in vitro DNA synthesis was analyzed with a polymerase-arrest assay in which prematurely terminated replication products were separated on a DNA sequencing gel. A synthetic DNA replication template was treated with increasing concentrations of chromium(III) chloride. The two lowest chromium doses used resulted in biologically relevant adduct levels (6 and 21 adducts per 1,000 DNA nucleotides) comparable with those measured in nuclear matrix DNA from cells treated with a 50% cytotoxic dose of sodium chromate in vivo. In vitro replication of the chromium-treated template DNA using the Sequenase version 2.0 T7 DNA polymerase (United States Biochemical Corp., Cleveland, OH) resulted in dose-dependent polymerase arrest beginning at the lowest adduct levels analyzed. The pattern of polymerase arrest remained consistent as chromium adduct levels increased, with the most intense arrest sites occurring 1 base upstream of guanine residues on the template strand. Replication by the DNA polymerase I large (Klenow) fragment as well as by unmodified T7 DNA polymerase also resulted in similar chromium-induced polymerase arrest. Interstrand cross-linking between complementary strands was detected in template DNA containing 62, 111, and 223 chromium adducts per 1,000 DNA nucleotides but not in template containing 6 or 21 adducts per 1,000 DNA nucleotides, in which arrest nevertheless did occur. Low-level, dose-dependent interstrand cross-linking between primer and template DNA, however, was detectable even at the lowest chromium dose analyzed. Since only 9% of chromium adducts resulted in polymerase arrest in this system, we hypothesized that arrest occurred when the enzyme encountered chromium-mediated interstrand DNA-DNA cross-links between either the template and a separate DNA molecule or the template and its complementary strand in the same molecule. These results suggest that the obstruction of DNA replication by chromium-mediated DNA-DNA cross-links is a potential mechanism of chromium-induced genotoxicity in vivo.

Intrauterine thoracentesis of fetal cystic lung malformations.

Fetal pulmonary malformations comprise a rare but often lethal group of congenital anomalies. Until recently, diagnosis and therapy were directed postnatally and therefore some cases of fetal compromise were inevitably missed. We present 2 cases in which intermittent thoracentesis of fetal cystic lung malformations resulted in a successful outcome. Intrauterine thoracentesis should be considered in the second and third trimester of pregnancy in cases which demonstrate early fetal compromise.