Lipid-induced mononuclear cell cytokine secretion in the development of metabolic aberration and androgen excess in polycystic ovary syndrome.

STUDY QUESTION: What is the effect of saturated fat ingestion on mononuclear cell (MNC) TNFα, IL-6 and IL-1ß secretion and circulating IL-6 levels in women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Women with PCOS exhibit increases in MNC-derived TNFα, IL-6 and IL-1ß secretion and circulating IL-6 following saturated fat ingestion even in the absence of obesity, and these increases are linked to metabolic aberration and androgen excess. WHAT IS KNOWN ALREADY: Cytokine excess and metabolic aberration is often present in PCOS. STUDY DESIGN, SIZE, DURATION: A cross-sectional design was used in this study of 38 reproductive-age women. PARTICIPANTS/MATERIALS, SETTING, METHODS: Groups of 19 reproductive-age women with PCOS (10 lean, 9 obese) and 19 ovulatory controls (10 lean, 9 obese) participated in this study that was performed at a tertiary academic medical centre. TNFα, IL-6 and IL-1ß secretion was measured from cultured MNC, and IL-6 was measured in plasma from blood sampling while fasting and 2, 3 and 5 h after saturated fat ingestion. Insulin sensitivity was determined using the Matsuda index following an oral glucose tolerance test. Androgen secretion was evaluated with blood sampling while fasting and 24, 48 and 72 h after an HCG injection. MAIN RESULTS AND THE ROLE OF CHANCE: Lean and obese women with PCOS exhibited lipid-induced incremental AUC increases in MNC-derived TNFα (489-611%), IL-6 (333-398%) and IL-1ß (560-695%) secretion and in plasma IL-6 levels (426-474%), in contrast with lean control subjects. In both PCOS groups, insulin sensitivity was lower (42-49%) and androgen secretion after HCG injection was greater (63-110%) compared with control subjects. The MNC-derived TNFα, IL-6 and IL-1ß and circulating IL-6 responses were inversely associated with insulin sensitivity and directly associated with fasting lipids and androgen secretion after HCG injection. LIMITATIONS, REASONS FOR CAUTION: The sample size of each of the four study groups was modest following group assignment of subjects by body mass. WIDER IMPLICATIONS OF THE FINDINGS: This study showcases the unique pro-inflammatory contribution of circulating MNC in the development of metabolic aberration and androgen excess in PCOS. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by grant R01 DK107605 to F.G. from the National Institutes of Health, the Indiana Clinical and Translational Sciences Institute Clinical Research Center which is funded in part by grant UL1TR002529 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award, and the Indiana University Center for Diabetes and Metabolic Diseases funded by grant P30 DK097512 from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. No conflicts of interest, financial or otherwise, are declared by the authors. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT01489319.

Assessment of myocardial metabolic flexibility and work efficiency in human type 2 diabetes using 16-[18F]fluoro-4-thiapalmitate, a novel PET fatty acid tracer.

Altered myocardial fuel selection likely underlies cardiac disease risk in diabetes, affecting oxygen demand and myocardial metabolic flexibility. We investigated myocardial fuel selection and metabolic flexibility in human type 2 diabetes mellitus (T2DM), using positron emission tomography to measure rates of myocardial fatty acid oxidation {16-[(18)F]fluoro-4-thia-palmitate (FTP)} and myocardial perfusion and total oxidation ([(11)C]acetate). Participants underwent paired studies under fasting conditions, comparing 3-h insulin + glucose euglycemic clamp conditions (120 mU·m(-2)·min(-1)) to 3-h saline infusion. Lean controls (n = 10) were compared with glycemically controlled volunteers with T2DM (n = 8). Insulin augmented heart rate, blood pressure, and stroke index in both groups (all P < 0.01) and significantly increased myocardial oxygen consumption (P = 0.04) and perfusion (P = 0.01) in both groups. Insulin suppressed available nonesterified fatty acids (P < 0.0001), but fatty acid concentrations were higher in T2DM under both conditions (P < 0.001). Insulin-induced suppression of fatty acid oxidation was seen in both groups (P < 0.0001). However, fatty acid oxidation rates were higher under both conditions in T2DM (P = 0.003). Myocardial work efficiency was lower in T2DM (P = 0.006) and decreased in both groups with the insulin-induced increase in work and shift in fuel utilization (P = 0.01). Augmented fatty acid oxidation is present under baseline and insulin-treated conditions in T2DM, with impaired insulin-induced shifts away from fatty acid oxidation. This is accompanied by reduced work efficiency, possibly due to greater oxygen consumption with fatty acid metabolism. These observations suggest that improved fatty acid suppression, or reductions in myocardial fatty acid uptake and retention, could be therapeutic targets to improve myocardial ischemia tolerance in T2DM.

Molecular evidence of prevalent dual piroplasma infections in North American raccoons (Procyon lotor).

Based on 18S rRNA sequence analyses 2 distinct genotypes of piroplasms have been described in raccoons. One genotype resides in the Babesia sensu stricto clade and the other in the Babesia microti-like clade. Since these organisms appear morphologically indistinguishable, it is unclear which strain is responsible for the majority of the infections in raccoons. In order to overcome these limitations we performed a molecular survey of raccoons using polymerase chain reaction assays specific for each genotype. We tested blood samples from 41 wild raccoons trapped in eastern North Carolina using PCR assays and found that 95% (39/41) had detectable piroplasm DNA. Ninety percent (37/41) of the samples contained Babesia sensu stricto DNA and 83% (34/41) samples contained Babesia microti-like DNA. DNA from both genotypes was present in 76% (31/41) samples suggesting a very high rate of co-infections. The presence of dual piroplasma infections in carnivores appears to be an uncommon finding. This study highlights the need for molecular assays for the accurate identification of piroplasma. Further studies are indicated to investigate the ability of these parasites to infect domestic animals as well as their zoonotic potential.

The identification of a genetically unique piroplasma in North American river otters (Lontra canadensis).

During a routine health check of a wild-caught North American river otter (Lontra canadensis) small piroplasms were noted within erythrocytes. Analyses of the 18S ribosomal ribonucleic acid (rRNA) gene sequences determined that this was a genetically unique organism most closely related to Babesia microti-like parasites found in other small carnivores. Subsequently 39 wild-trapped North American river otters from North Carolina were tested for the presence of piroplasma deoxyribonucleic acid (DNA) via polymerase chain reaction and piroplasma DNA was detected in 82% (32/39) of these samples. Sequencing of partial 18S rRNA genes from selected cases determined that they were identical to the sentinel case. This report documents the existence of a genetically unique piroplasma in North American river otters and indicates that the prevalence of piroplasma in North Carolina otters is quite high. The pathogenic potential of this organism for otters or other species remains unknown.

Immunologic responses and adverse reactions to Freund's-adjuvanted porcine zona pellucida immuno-contraceptives in domestic cats.

A vaccine of native PZP with Freund's adjuvant has been widely used in zoo and wild ungulates, but safety in felids has not been evaluated. General health, immune response, and ovarian histology were assessed in five domestic cats vaccinated with PZP-Freund's and five cats given Freund's adjuvant alone. Peak antibody titers occurred 3 weeks after the third vaccination, and no ovarian lesions were present 6 months after vaccination. Seven cats developed extensive granulomatous reactions at injection sites, lymph nodes, and multiple visceral organs including lungs and brain. Persistent hypercalcemia and compromised renal function occurred in three cats with elevated serum calcitriol of probable macrophage origin. One cat died from an injection site sarcoma. Because of these severe adverse reactions, Freund's adjuvant is contraindicated in cats, and other adjuvants for PZP vaccines should be tested in cats for adverse reactions before use.

Survey of necropsy results in captive red wolves (Canis rufus), 1992-1996.

Through the Red Wolf Species Survival Plan, the captive red wolf (Canis rufus) population was developed with the intent of reestablishing wild populations. One part of the plan was a survey for diseases that might occur as a result of population homogeneity or that might impede breeding success and reintroduction. For this survey, complete necropsies and histopathologic analyses were performed on 62 red wolves from 1992 to 1996. Major causes of 22 neonatal deaths were parental trauma, parasitic pneumonia, and septicemia. Common neonatal lesions included pododermatitis and systemic ascariasis. Cardiovascular anomalies and systemic parasitism were found in two juveniles. Causes of death in the 38 adults included conspecific trauma, neoplasia, or gastrointestinal diseases such as necrotizing enteritis, intestinal perforation, and gastric volvulus. Lymphosarcoma represented 50% of the fatal neoplasms. Three adults died from cardiovascular failure or hyperthermia during handling, and several adults were euthanized for suspected genetic diseases. Overall, the captive population had few significant health problems, but population fitness might be improved by continued removal of potentially deleterious genes from the breeding population and by modifying the husbandry of neonates and adults.

Chromosome aberrations in cultured central mudminnow heart cells and Chinese hamster ovary cells exposed to polycyclic aromatic hydrocarbons and sediment extracts.

1. Genotoxicity experiments were conducted with cultured fish cells to determine if the high frequency of epidermal papillomas observed in lemon sole from Sturgeon Bank, where a sewage treatment plant discharges, could be correlated with contamination of the sediments with chemicals such as 3,4-benzopyrene. 2. The frequency of chromosome aberrations was measured in cultured Umbra limi heart (U1-H) and Chinese hamster ovary (CHO) cells following exposure to the polycyclic aromatic hydrocarbons (PAH) 3,4-benzopyrene (BP), 1,2,5,6-dibenzanthracene (DBA), 1,2-benzanthracene (BA), and pyrene (PY), activated using S9 prepared from rainbow trout liver. 3. An increase in the chromosome aberration frequency was only observed following exposure to fish S9-activated BP in both cell lines. 4. Following exposure of the cells to both Sturgeon Bank and Spanish Bank sediment extracts, it was determined that a higher level of toxic and genotoxic activity was associated with the Sturgeon Bank sediments. 5. Since the detection of PAH genotoxicity requires the presence of S9, and since a higher level of genotoxic activity was noted following sediment extract exposures with no S9 present, this suggests that the extracts contain a complex mix of chemicals, some of which express genotoxic activity. 6. An assessment using the micronucleus test failed to indicate in vivo genotoxicity in fish collected from Sturgeon and Spanish Banks. 7. It was, therefore, difficult to associate the observed sediment genotoxicity with the previously noted high incidence of epidermal papillomas in lemon sole from this area.

DNA repair synthesis in cultured fish and human cells exposed to fish S9-activated aromatic hydrocarbons.

Unscheduled DNA repair synthesis was measured autoradiographically in cultured rainbow trout gonad (RTG) and human fibroblast (HF) cells following exposure to aflatoxin B1 (AFB1), 3,4-benzopyrene (BP), 1,2,5,6-dibenzanthracene (DBA), 1,2-benzanthracene (BA) and pyrene (PY) activated with S9 prepared from rainbow trout liver. S9 from rainbow trout injected with Arochlor 1254 or an oil extract was compared with S9 from Fischer rats injected with Arochlor 1254 for the ability to activate AFB1 and cause DNA repair in RTG and HF cells. All three types of S9 activated AFB1, but the measured DNA repair response was greater in the HF cells. A significant grain count response was found following exposure of HF cells to fish S9-activated BP. Using assay conditions which enhance fish cell grain counts, a significant level of DNA repair was also found in RTG cells exposed to fish S9-activated BP. Marginal but statistically significant amounts of DNA repair were elicited in HF and RTG cells exposed to rainbow trout S9-activated BA and DBA, but no response was detected following PY exposure. Fish S9 was found to be able to activate a series of polycyclic aromatic hydrocarbons (PAH) and cause DNA repair synthesis in both fish and mammalian cells. The magnitude of the repair response roughly parallels the carcinogenic potential of the PAHs. These results elicit trans species and phyla comparisons which help to validate fish as models for aquatic carcinogenesis research, and also demonstrate PAH DNA-damaging effects on fish DNA, adding further credence for studying the effects of these chemicals on aquatic organisms.

Comparison of DNA-repair synthesis, chromosome aberrations and induction of micronuclei in cultured human fibroblasts, Chinese hamster ovary and central mudminnow (Umbra limi) cells exposed to chemical mutagens.

In mammalian cells it has previously been observed that low DNA-repair activity is correlated with high chromosome-aberration frequency. Since fish cells typically express comparatively low amounts of DNA repair, the chromosome aberration test holds potential as a sensitive fish genotoxicity assay. A comparison of in vitro DNA-repair activity showed HF greater than CHO greater than Ul-H = Ul-F following exposure to MNNG and 4NQO. Although peak chromosome-aberration frequency varied CHO greater than Ul-H greater than HF, at comparable mutagen concentrations the relationship was Ul-H greater than HF greater than CHO following 4NQO exposure and Ul-H greater than HF = CHO after MNNG exposure. Analyzing for chromosome aberrations at high mutagen concentrations was not possible due to mitotic inhibition/toxicity which varied according to the mutagen and cell line. Micronuclei frequency varied CHO greater than Ul-H greater than HF = Ul-F. In CHO and Ul-H, a 10-15-fold increase over controls compares with only a 2-3-fold increase for HF and Ul-F. These differences are likely related, in part, to the cell-division rate of each line and the coincident repair of the damaged DNA. Reasons for the lack of negative correlation between DNA repair and chromosomal damage in fish cells are discussed.

DNA repair synthesis following exposure to chemical mutagens in primary liver, stomach, and intestinal cells isolated from rainbow trout.

DNA repair synthesis was autoradiographically measured in liver, stomach, and intestinal cells isolated from rainbow trout which were exposed in vitro to the chemical mutagens, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitroquinoline 1-oxide, and aflatoxin B1. The level of repair was greatest in primary hepatocytes which responded to all three mutagens. Only nominal amounts of repair were detected in stomach cells following N-methyl-N'-nitro-N-nitrosoguanidine and 4-nitroquinoline 1-oxide exposures and in intestinal cells following 4-nitroquinoline 1-oxide exposure. In comparison with cultured rainbow trout cells, the quantity of DNA repair found in primary cells is significantly less.

DNA repair synthesis in cultured mammalian and fish cells following exposure to chemical mutagens.

Unscheduled DNA repair synthesis (UDS) was measured autoradiographically in HF, CHO, RTG, RTO, CH and FHM cells given a 3-h exposure to MNNG, 4NQO, NA2AAF and AFB1. All the chemicals produced a dose-response, the magnitude of which varied with the particular chemical and cell line. HF produced the greatest response, CHO less and the fish cell lines the least. The response of all fish cell lines was approximately equal for a particular chemical. A number of factors were investigated to account for the comparative differences in UDS response. The time course of repair in HF, CHO and RTG following a 3-h exposure to MNNG or 4NQO was the same. As S-phase nuclei were observed in control slides and the amount of repair following UV exposure varies HF greater than CHO greater than RTG, neither 3HTdR nor mutagen uptake is limiting. The observed results are discussed.

Mutagenicity of fecal extracts from carnivorous and herbivorous animals.

Extracts of the feces of 3 carnivorous animals (dog, river otter and sea gull) and 5 herbivorous animals (cow, horse, sheep, chicken and goose) induced chromosome aberrations (breaks and exchanges) in cultured CHO cells. The addition of CuII (10(-4)M) enhanced the clastogenic effect of fecal extracts of the examined animals with the exceptiion of 1 dog and 3 cow samples. Catalase reduced the chromosome-breaking and mitosis-inhibiting capacities of fecal extracts. These results indicate the presence of hydrogen peroxide-forming compounds. The possibility must be considered that animal and human excreta may be a major source of mutagens entering man's environment.

Can mutation theories of carcinogenesis set priorities for carcinogen testing programs?

The recent activity in designing, validating and implementing short-term tests for carcinogens has been spurred by the fairly convincing correlation between the carcinogenicity and mutagenicity of chemicals and by the assumption that mutations are somehow involved in neoplastic transformation. Moreover, it has been tacitly assumed that the mutagenic capacity alone of compounds would induce regulatory agencies to pass rules for their removal from man's environment, and would lead the public to avoid them. The actual response, however, is quite different. Government departments shy away from making any decisions on the basis of in vitro test systems, the public at large is becoming irritated by daily announcements that many of their cherished habits could adversely affect their health, and industries feel threatened and may reduce their search for new beneficial chemicals. The reluctance to accept wholeheartedly the mutagenicity tests for the detection of carcinogens is partly due to the uncertainty about the involvement of mutations in the formation of benign and malignant tumors. Following the initial rapid advances in the detection of environmental chemicals with carcinogenic and mutagenic properties, we seem to have arrived at the cross roads: we must now set new priorities for future research, and must make an unbiased assessment of the actual hazard of a compound to man and the human population.