Reactive oxygen species, diabetes and toxicity in the placenta - a workshop report.

The placenta, in addition to its myriad of functions during development, is recognized as a target for the toxic actions of chemicals. Presentations in this workshop summarized the state of the science with respect to drug metabolizing enzyme expression and activity as well as drug transporter protein expression. Chemical induction of reactive oxygen species (ROS) formation was presented as a unifying mechanism potentially important in the development of teratogenesis, postnatal cancers, and diabetes.

Benzo(a)pyrene increases phosphorylation of p53 at serine 392 in relation to p53 induction and cell death in MCF-7 cells.

Although benzo(a)pyrene (BP) induces apoptosis in vitro in murine Hepa1c1c7 cells and in vivo indications of apoptosis in rat lung exist, related cellular mechanisms in human cells are not known. p53 protein participates in several apoptotic processes. We found that BP induces cell death in human MCF-7 breast adenocarcinoma cells at 48 and 72h but not in human A549 lung carcinoma cells. BP did not induce measurable caspase-3-like protease activity or internucleosomal DNA fragmentation in either cell types. However, procaspase-7 cleavage in MCF-7 cells by BP-treatment indicates activation of caspase-7 meaning that apoptosis is most likely involved in BP-induced MCF-7 cell death. BP-7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts and level of p53 protein increased dose-dependently, but more extensively in MCF-7 cells. Phosphorylation of p53 protein at serines 15, 20, 46 and 392 increased in MCF-7 cells. Increase in phosphorylation at serine 392 was clear already at 24h by 1 microM concentration of BP. Increase of phosphorylation at other sites occurred only with higher concentrations or at later time points in relation to the increase of p53 protein. These results suggest that serine 392 phosphorylation is the first stabilizing event of p53 associated with BP exposure and subsequent cell death in MCF-7 cells.

Quantitative estimation of mercury intake by toxicokinetic modelling based on total mercury levels in humans.

Mercury is a toxic metal that can be disseminated into the environment from both natural and anthropogenic sources. Human exposure to the metal stems mainly from food, and more particularly from the consumption of fish and other seafoods. Examining dietary exposure and measuring mercury levels in body tissues are two ways of estimating exposure to mercury. In this study, we utilized a modelling system consisting of three linear toxicokinetic models for describing the fate of methyl mercury, inorganic mercury, and metallic mercury in the body, in order to estimate daily intake of mercury as measured through total mercury concentrations in the blood. We then compared the results stemming from our modelling system to those of the detailed semi-quantitative food frequency questionnaire (FFQ) of the Norwegian Fish and Game (NFG) Study, a project that focused on dietary mercury exposure. The results indicate that toxicokinetic modelling based on blood levels gave higher daily intake values of mercury compared to those of the FFQ. Furthermore, the former had a wider range of estimates than the latter. The properties of the toxicokinetic model or limitations in the dietary exposure assessment could be posited as reasons for the differences between the respective methods. Moreover, the results may have been influenced by sources of mercury exposure that cannot be described as dietary, such as amalgam fillings.

DNA damage caused by benzo(a)pyrene in MCF-7 cells is increased by verapamil, probenecid and PSC833.

The aim of this study was to clarify whether pharmaceutical drugs capable of inhibiting ABC-transporters affect the toxicity of benzo(a)pyrene (BP). MCF-7 breast adenocarcinoma cells were cultured for 24 and 48 h with benzo(a)pyrene (1 microM) and the transporter inhibitors verapamil (0.125-100 microM), PSC833 (0.05-5 microM) or probenecid (0.05-2 mM). DNA binding of benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) was analyzed by synchronous fluorescence spectrophotometry and p53 protein by immunoblotting. BP metabolism was studied using thin layer chromatography (TLC). MTT assay and ATP quantitation were used for the analysis of cell viability. At 24 h there was no statistically significant increase in the DNA-adduct formation by any of the used inhibitors. However, at 48 h all of the inhibitors, in concentrations known to effectively block ABC transporters, increased the BPDE-DNA adduct formation 1.5 to 2-fold compared to adduct formation with BP only. PSC833 and verapamil also increased p53 protein expression at 48 h (p<0.05). Probenecid decreased glucuronidation of (3)H-BP metabolites. Other inhibitors did not decrease statistically significantly the overall formation of water-soluble metabolites. BP alone slightly decreased viability of cells at 48 h according to ATP quantitation as compared to vehicle treated controls (86.4+/-16.4%). Even though the used inhibitors showed some cytotoxicity, the combination of BP and inhibitors did not decrease cell viability in synergistic manner. According to these results certain pharmaceutical drugs may increase DNA damage caused by benzo(a)pyrene in MCF-7 cells at least partly through the inhibition of transporters. Taking into account the complex metabolism of BP and lack of specificity of the inhibitors used, it is likely that increased DNA damage seen in this study was the result of multiple interactions between the inhibitors, BP metabolism and the efflux of the compounds.

The xenoestrogens, bisphenol A and para-nonylphenol, decrease the expression of the ABCG2 transporter protein in human term placental explant cultures.

Many endogenous and xenobiotic compounds are substrates and regulators of human placental ABC transporters. ABCG2 is protecting fetus against foreign chemicals. Environmental xenoestrogens, like bisphenol A (BPA) and p-nonylphenol (p-NP), mimic natural estrogens and can affect hormonal systems. Effects of BPA, p-NP, DES (diethylstilbestrol) and estradiol (E2), on ABCG2 expression were studied using human first trimester and term placental explants. Role of estrogen receptors (ER) in the effects of chemicals was studied by ER antagonist. Term placenta expressed less ABCG2 protein. In term placentas BPA (p < 0.05), p-NP (p < 0.01) and E2 (p < 0.05) decreased the ABCG2 protein expression after 48 h exposure while after 24 h exposure, only E2 decreased the expression (p < 0.05). The chemicals did not affect ABCG2 in first trimester placentas. The ER antagonist affected differently the responses of chemicals. In conclusion, environmental xenoestrogens downregulate placental ABCG2 protein expression depending on gestational age.

Human placenta: a human organ for developmental toxicology research and biomonitoring.

Pregnant mothers are exposed to a wide variety of foreign chemicals. This exposure is most commonly due to maternal medication, lifestyle factors, such as smoking, drug abuse, and alcohol consumption, or occupational and environmental sources. Foreign compounds may interfere with placental functions at many levels e.g. signaling, production and release of hormones and enzymes, transport of nutrients and waste products, implantation, cellular growth and maturation, and finally, at the terminal phase of placental life, i.e. delivery. Placental responses may also be due to pharmaco-/toxicodynamic responses to foreign chemicals, e.g. hypoxia. On the other hand, placental xenobiotic-metabolizing enzymes can detoxify or activate foreign chemicals, and transporters either enhance or prevent cellular accumulation and transfer across the placenta. The understanding of what xenobiotics do to the placenta and what the placenta does to the xenobiotics should provide the basis for the use of placenta as a tool to investigate and predict some aspects of developmental toxicity. This review aims to give an update of the fate and behavior of xenobiotics in the placenta from the viewpoint of xenobiotic-metabolizing enzymes and transporters. Their response levels will be described according to gestational status and methods used. The effects of foreign chemicals on placental metabolizing enzymes will be discussed. Also, interactions in the transporter protein level will be covered. The role of the placenta in contributing to developmental effects and fetotoxicity will be examined. The toxicological effects of maternal medications, smoking, and environmental exposures (dioxins, pesticides) as well as some possibilities for biomonitoring will be highlighted.

Organic anion transporter 4 (OAT 4) modifies placental transfer of perfluorinated alkyl acids PFOS and PFOA in human placental ex vivo perfusion system.

INTRODUCTION: Perfluorinated alkyl acids (PFAAs) are widely used in industry and consumer products. Pregnant women are exposed to PFAAs and their presence in umbilical cord blood represents fetal exposure. Interestingly, PFAAs are substrates for organic anion transporters (OAT) of which OAT4 is expressed in human placenta. METHODS: To evaluate the contribution of OAT4 and ATP-binding cassette transporter G2 (ABCG2) proteins in the transplacental transfer of perfluoro octane sulfonate (PFOS) and perfluoro octanoate (PFOA) an ex vivo dual recirculating human placental perfusion was used. Altogether 8 placentas from healthy mothers with uncomplicated pregnancies were successfully perfused. RESULTS: Both PFOS and PFOA crossed the placenta as suggested by in vivo data in the literature. The expression of OAT4 and ABCG2 proteins were studied by immunoblotting and correlation with the transfer index %(TI %) of PFOS and PFOA at 120 and 240 min (n = 4) was studied. The expression of OAT4 was in negative correlation with TI % of PFOA (R(2) = 0.92, p = 0.043) and PFOS (R(2) = 0.99, p = 0.007) at 120 min while at 240 min the correlation was statistically significant only with PFOA. The expression of ABCG2 did not correlate with TI% of PFOS or PFOA. DISCUSSION: Data obtained in this study suggest the involvement of OAT4 in placental passage of PFAAs. Placental passage of PFOS and PFOA is modified by the transporter protein OAT4 but not by ABCG2. This is the first study indicating that OAT4 may decrease the fetal exposure to PFAAs and protect the fetus after maternal exposure to PFAAs but further studies are needed to confirm our findings.

Criteria and challenges of the human placental perfusion ­ Data from a large series of perfusions.

Perfusion of human placental cotyledon has been used extensively to study transplacental transfer of endogenous and exogenous compounds. However, many challenges in the use of the method exist, including availability of placentas and complexity of the method itself. In Kuopio, Finland we have carried out human placental perfusions since 2005 using the same method with data now from over one hundred perfusions. This has allowed us to study whether the way of delivery, placental weight, and/or the length of pregnancy affect the two major criteria of a successful perfusion: volume loss (leak) from fetal to maternal circulation, and transplacental transfer of the reference compound antipyrine. The only statistically significant result was the reduction of the fetomaternal ratio of antipyrine by the placental age over 40 weeks (p=0.0004). The success criteria were not affected by the weight of the placenta or the way of delivery. There was no effect by the antipyrine concentration on antipyrine transfer. In vitro incubation with different concentrations of study compounds and different tubing materials could offer an easy way to study potentially reduced recovery due to binding to perfusion system.

High density lipoprotein and apolipoprotein A-i during physical inactivity. Demonstration at low levels in patients with spine fracture.

Lack of physical activity appears to have deleterious effects on serum lipoproteins. Twenty-three patients who were completely immobilised by traumatic fracture of the spine had significantly lower (P < 0.001) plasma high density lipoprotein cholesterol (HDL-C) and apolipoprotein A-I levels than normally mobile paired control subjects. The low density lipoprotein cholesterol (LDL-C) levels of the immobile patients were not different from those of controls but the LDL triglyceride (LDL-TG) of the patients was increased. The patients had a significantly higher LDL/HDL-C ratio and HDL-C/apoprotein A-I ratio than the controls. These results suggest that the increased risk of ischemic heart disease in physically inactive people is partially accounted for by low plasma HDL levels. On the other hand, caution is needed in the interpretation of HDL findings in clinical conditions where the physical activity of the patients is limited.

Aseptic necrosis of the femoral head during pregnancy.

Two patients are presented with rare aseptic osteonecrosis of the femoral head appearing during the last trimester of pregnancy. Both patients suffered from increasing pain in one hip joint, beginning about one month before parturition. Radiographs demonstrated local osteoporotic changes of the femoral head on the painful side. Needle aspiration of the hip joint yielded a scant amount of clear, sterile synovial fluid and gave prompt relief of pain in both cases. The patients' history did not demonstrate any predisposing factors to osteonecrosis other than pregnancy. Treatment comprised omission of weight-bearing on the affected lower extremity for a month or two. The patients had completely recovered one year postpartum, and radiographs were normal.

An examination of whether human placental perfusion allows accurate prediction of placental drug transport: studies with diazepam.

INTRODUCTION: Presently, no well-validated predictive tools are available for human placental transfer. We studied the transplacental passage of diazepam (DZP) in a recirculating dual human placental perfusion and compared the data with in vivo clinical data from the literature. METHODS: Term placentas from healthy mothers without medication were used. The dual, recirculating perfusion technique was used. DZP (2 microg/ml, n = 4; 200 ng/ml, n = 3) and the reference compound antipyrine (100 microg/ml) were added into the maternal circulation simultaneously. The disappearance of drugs from the maternal circulation and appearance into the fetal circulation were followed every 15 min for 2 h. RESULTS: DZP was detectable in the fetal circulation within 15 min in all of the perfusions indicating rapid transfer. DZP concentrations in the maternal circulation were higher than in the fetal circulation throughout the perfusion with both initial concentrations. At the end of the perfusion, the feto-maternal ratio was 0.48 +/- 0.11 (mean +/- S.D.) and the transfer from the maternal to the fetal compartment 18.4 +/- 3.6% with 2 microg/ml of DZP and 0.55 +/- 0.10 and 20.5 +/- 3.1% with 200 ng/ml of DZP, respectively. DZP concentrations in the perfused area of the placenta were in average 2 times higher than in the maternal perfusate and 3.6 times higher than in the fetal perfusate. Total recovery of DZP from samples, perfusion fluid, and perfused tissue was 37.6 +/- 21%. DISCUSSION: Since animal studies in vivo do not accurately predict human placental transfer and it is problematic to study placental transfer of drugs in humans in vivo, the present human placental perfusion system could serve as one part of a test battery for fetotoxicity. However, although our earlier studies and those from the literature indicate a good correlation between in vivo and placental perfusion data, the present study shows this is not the case for all drugs.

Chronic unilateral low-back pain. Predictors of outcome of facet joint injections.

One hundred nine patients with chronic (3-36 months; mean, 13.4 months) unilateral low-back pain and no signs of sciatica were subjected to facet joint injection, randomized in three therapy groups: cortisone and local anesthetic injected intra-articularly, the same mixture injected pericapsularly, and physiologic sodium hydrochloride injected intra-articularly into two facet joints. To evaluate the results, three outcome variables were formed: work, subjective, and disability outcome. The inappropriate signs (IAS) recorded before injections had the best predictability for a good outcome. The mode of injection or duration of symptoms had no significance as a predictor. It was concluded that the outcome after facet joint injection depends on the patient's biopsychosocial chances of self-facilitated improvement. If abnormal illness behavior and distress are found, it helps to estimate the response for treatment and to choose a realistic method of treatment.

Reduction of bone retropulsed into the spinal canal in thoracolumbar vertebral body compression burst fractures. A prospective randomized comparative study between Harrington rods and two transpedicular devices.

STUDY DESIGN: This was a prospective, randomized study. OBJECTIVE: To compare the ability of three methods of internal fixation (Harrington rods, AO internal fixator, posterior segmental fixator) to obtain reduction of intracanal fragments in thoracolumbar vertebral compression burst fractures. SUMMARY OF BACKGROUND DATA: Sixty-seven acute thoracolumbar compression burst fractures of T12 or L1 were randomized into three groups that were treated using one of the three methods. Reduction was accomplished indirectly by distraction applied using the fixation device. METHODS: The spinal canal encroachment was calculated as a percentage of the estimated pre-injury value from serial transverse computed tomographic scans obtained on admission and immediately after surgery. RESULTS: The median preoperative sagittal encroachment of the spinal canal was 37% (range, 0-90%) of the normal diameter. All three methods of internal fixation produced a spinal canal clearance provided that the patient was operated on within 4 days after trauma. The median postoperative encroachment varied from 13% (range, 0-37%) to 22% (range, 0-37%), the best reduction being attained using Harrington rods and the poorest with the posterior segmental fixator. There was a suggestive statistical significance between these two. CONCLUSION: The differences in postoperative spinal canal encroachment and ability to obtain spinal canal clearance observed between the devices studied were small. There seems to be no reason to base the choice of the operative method in thoracolumbar fractures on any hypothetical differences in reductive power between Harrington rods and the AO internal fixator.

A controlled immunohistochemical study of inflammatory cells in disc herniation tissue.

STUDY DESIGN: The presence and abundance of inflammatory cells was studied immunocytochemically in lumbar disc herniations (DH) and macroscopically normal discs for comparison. OBJECTIVES: The objective of the study was to characterize inflammatory cells that appear in herniated disc tissue and to study the relative abundance of various types of inflammatory cells. SUMMARY OF BACKGROUND DATA: Only few macrophages were observed in control discs, whereas abundant macrophages were present in half of the DH. Other types of inflammatory cells were less often abundant in the present material. In about a third of the DH interleukin-1 beta-expressing cells were also observed. METHODS: Twenty-four DH and control tissue from five discs were studied immunocytochemically, using specific monoclonal antibodies to various types of inflammatory cells and interleukin-1 beta. The results were compared with corresponding clinical data. Macrophages were studied with an antibody to CD68 antigen and Ber-MAC3 antibody separately. RESULTS: The obtained results suggest a variable inflammatory cell response in DH, which seems to be often dominated by macrophages at the time of operation. Thus previous suggestions of sometimes very active inflammation in DH tissue are supported. CONCLUSIONS: Inflammation may be important in disc tissue pathophysiology, possibly also in discogenic pain mechanisms.

Survival after massive transfusions exceeding four blood volumes in patients with blunt injuries.

The authors' experience with 29 patients with blunt injuries who sustained massive transfusions exceeding four blood volumes in the initial posttraumatic 12 hours was reviewed. The overall mortality was 62 per cent. Only one patient survived when the amount of transfusions exceeded 50 units. The most common bleeding sites were abdominal and pelvic. Half of the dead succumbed within the first 24 hours. Of those who died, seven died from brain injury, six from multiple organ failure (MOF), and five from uncontrollable bleeding. Initial shock was observed in 55 per cent of patients. The high mortality of patients with massive transfusions and MOF related to the duration of shock strongly promote the importance of initial volume resuscitation.

Complications of transpedicular lumbosacral fixation for non-traumatic disorders.

We analysed the complications encountered in 102 consecutive patients who had posterolateral lumbosacral fusion performed with transpedicular screw and rod fixation for non-traumatic disorders after a minimum of two years. Of these, 40 had spondylolysis and spondylolisthesis, 42 a degenerative disorder, 14 instability after previous laminectomy and decompression, and six pain after nonunion of previous attempts at spinal fusion without internal fixation. There were 75 multilevel and 27 single-level fusions. There were 76 individual complications in 48 patients, and none in the other 54. The complications seen were screw misplacement, coupling failure of the device, wound infection, nonunion, permanent neural injury, and loosening, bending and breakage of screws. Screw breakage or loosening was more common in patients with multilevel fusions (p < 0.001). Screws of 5 mm diameter should not be used for sacral fixation. Forty-six patients had at least one further operation for one or several complications, including 20 fusion procedures for nonunion. The high incidence of complications is a disadvantage of this technically-demanding method.

Anterolateral decompression for neural involvement in thoracolumbar fractures. A review of 78 cases.

Of a total of 905 patients with fracture or fracture-dislocation of the thoracolumbar spine admitted from 1969 to 1982, a neurological deficit was present in 334 (37%). All unstable injuries were initially treated by reduction and posterior fusion. In 79 of these patients, an anterolateral decompression was undertaken later because of persistent neurological deficit and radiographic demonstration of encroachment on the spinal canal. One patient died of pulmonary embolism; 78 were reviewed after a mean period of four years. Of these 78 patients 18 made a complete neurological recovery while 53 appeared to have benefited from the procedure; 25 remained unchanged. The best results were obtained in burst fractures at thoracolumbar and lumbar levels when a solitary detached fragment of a vertebral body had been displaced into the spinal canal. These results indicate that anterolateral decompression of the spinal canal should be considered, after careful evaluation, for certain injuries of the spine in which there is severe neural involvement.

Lumbar facet joint syndrome. A randomised clinical trial.

A group of 109 patients with unilateral low back pain for over three months were randomised to receive one of three types of injection treatment: cortisone and local anaesthetic injected into two facet joints (28), the same mixture around two facet joints (39), or physiological saline into two facet joints (42). The effect of the treatment was evaluated in relation to work attendance, pain, disability and movements of the lumbar spine. Patients were examined one hour and two and six weeks after treatment and also completed a questionnaire after three months. A significant improvement was observed in work attendance, pain and disability scores, but this was independent of the treatment given and movements of the lumbar spine were not improved. Of the 70 patients with initial pain relief after injection, 36% reported persisting benefit at the three month follow-up, independent of the mode of treatment given. We conclude that facet joint injection is a non-specific method of treatment and the good results depend on a tendency to spontaneous regression and to the psychosocial aspects of back pain.

Factors associated with nonunion in conservatively-treated type-II fractures of the odontoid process.

In type-II fractures of the odontoid process, the treatment is either conservative in a halo vest or primary surgical stabilisation. Since nonunion, requiring prolonged immobilisation or late surgery, is common in patients treated in a halo vest, the identification of those in whom this treatment is likely to fail is important. We reviewed the data of 69 patients with acute type-II fractures of the odontoid process treated in a halo vest. The mean follow-up was 12 months. Conservative treatment was successful, resulting in bony union in 32 (46%) patients. Anterior dislocation, gender and age were unrelated to nonunion. However, nonunion did correlate with a fracture gap (> 1 mm), posterior displacement (> 5 mm), delayed start of treatment (> 4 days) and posterior redisplacement (> 2 mm). We conclude that patients presenting with these risk factors are unlikely to achieve bony union by treatment in a halo vest. They deserve careful attention during the follow-up period and should also be considered as candidates for primary surgical stabilisation.

Microsomal metabolism of carbamazepine and oxcarbazepine in liver and placenta.

Metabolism of both carbamazepine (CBZ) and oxcarbazepine (OCBZ) were catalyzed by human liver microsomes and microsomes from livers of CBZ-induced or non-induced C57BL/6 mice. Human placental microsomes metabolized only OCBZ. Mouse liver microsomes metabolized CBZ to carbamazepine-10,11-epoxide (CBZ-E), 10-hydroxy-10,11-dihydro-carbamazepine (10-OH-CBZ), 3hydroxy-carbamazepine (3-OH-CBZ), 10,11-trans-dihydroxy-10,11-dihydro-carbamazepine (10,11-D) and to an unidentified metabolite. CBZ-pretreatment of mice increased both ethoxyresorufin O-deethylase activity in the liver and the amount of CBZ-E in microsomal incubations regardless of the age of mice. Human liver microsomes catalyzed the formation of CBZ to 9-hydroxymethyl-10-carbamoyl acridan (9-AC) in addition to CBZ-E, 3-OH-CBZ and 10-OH-CBZ. OCBZ was metabolized to its active metabolite in all incubations. An unknown metabolite was also present in some of the incubations. Human liver microsomes catalyzed only minute covalent binding of CBZ and OCBZ to DNA. Binding of OCBZ was, however, one order of magnitude greater than binding of CBZ. Human placental microsomes from the mothers on CBZ therapy did not catalyze CBZ metabolism. The same microsomes catalyzed OCBZ metabolism to 10-OH-CBZ and to an unknown metabolite. These results indicate autoinduction in CBZ metabolism in mouse liver. Due to the higher binding of OCBZ than CBZ to DNA in vitro, further studies on the potential mutagenicity of OCBZ may be warranted.