FISH Abnormalities Research Articles

FISH- Target Gene Panel Profile for Chronic Lymphocytic Leukemia/Small Cell Lymphocytic Lymphoma [CLL/SLL

CLL/SLL test data for target FISH 4 probe (Vysis Inc) panel comprising of D13S319 (13q14.3)/LSI 13q34, LSI p53 (17p13.1), LSI ATM (11q22.3) to detect deletions (del) and CEP 12 to detect trisomy on the majority of cases and a 3 probe panel: MYB LSI 6q22–23 (homebrew), LSI ATM (11q22.3), and CEP 12 on 59 cases, along with flow cytometry, and cytogenetics were retrospectively analyzed.A total of 888 CLL/SLL FISH tests were ordered, 509 [Av age 67.7yrs, <50 yrs = 36; M:F ratio 319:190; Specimen type: 239 Blood, 252 Bone marrow, 18 tissue] had CLL/SLL immunophenotype [light chain restricted B-cells coexpressing CD5 and CD19 with CD23], 59 had a differential diagnosis based on surface markers, 137 were polyclonal, normal immunophenotype or too few B-cells etc. and 124 had only a FISH test.Among CLL/SLL flow confirmed cases 349 [349/509=68.6%] were abnormal for 4- probe panel FISH: 160 del 13q34 [45.8 % (122-del 13, 17-biallelic del 13, 21-mono/biallelic del)], 71 extra copy of 12 [20.3%], 17 del ATM [5%], 12 del p53 [3.4%], and 89 with ≥2 FISH abnormalities [25.5%]. Concurrent cytogenetic results obtained in 151 cases were abnormal in 43 [28.5%]. The FISH results in 108 normal karyotype cases were: 39 normal, 1 del ATM [in 20% cells], 4 del p53 [11–29%], 14 extra copy of 12 [14–79%], 35 del 13q34 [13–98%], 15 ≥ 2 FISH abnormalities. FISH results in 43 abnormal karyotype cases: a normal FISH result in 10 [1- balanced translocation, 3- loss of Y, 2-del 6q, 1-complex karyotype with del 6q, one t(8;14), 1-trisomy 8, and 1-t(13;15)(q14;q26)]. An extra copy of 12 by FISH had concordant +12 karyotype in 2 cases, loss of Y in 3, trisomy 15 in one, 2 had multiple changes with +12, 1 had multiple changes without +12. In 8 del 13q34 FISH cases, karyotypes showed a loss of Y in 4, concordant del 13q in one, multiple abnormalities not involving 13 in 2 cases of CLL with increased myeloblasts [9–10%] by flow were del 5q, del 7q and del 20q in one and deletions of 5q, 9q and 18q in the other. A del p53 FISH case showed a loss of 17 in a 3n+ karyotype. Of 15 cases with ≥ 2 FISH abnormalities, the karyotypes had partial concordance in 8, discordant in 6 and concordant in 1.One of 59 [1.6%] tested with 3-probe panel had a del MYB [6q22–23] and 6 [6/151=4%] flow confirmed CLL with normal or multiple probe abnormalities for 4-probe panel had a cytogenetic del 6q. Of 10 cases considered as T-CLD 4 had ATM deletion and 6 were normal by FISH. In 14 CD23 negative cases IgH/BCL1 FISH probe detected t(11;14) and confirmed mantle cell lymphoma. Three cases were diagnosed with PLL, one had a deletion of p53 and an abnormal karyotype, two were normal by FISH. Multiple probes [Vysis Inc]/panels which included the IgH probe were ordered in 57 cases with CLL immunophenoype, 10 cases had an uncommon IgH rearrangement not included in the panels.Because of the greater efficacy of FISH compared to karyotyping which is limited by difficulties in culturing lymphoid versus myeloid cells, chromosome analysis can be considered in conjunction with FISH for CLL. Although the 4-FISH probe panel stratified CLL by prognosis in 68.6 % cases, the addition of del 6q probe would enhance identifying cases with poor prognosis. Since IgH rearrangements appear to be prevalent, a 3 color IgH/BCL1 probe would increase the strength of the CLL panel by identifying any IgH rearrangement [usually associated with a more aggressive disease] and also provide a differential diagnosis, particularly, in CD23 negative cases.

Utility of Interphase FISH To Stratify Patients into Cytogenetic Risk Categories at Diagnosis of AML in an ECOG Clinical Trial (E1900).

Background: Cytogenetic risk categories based on conventional chromosome studies are widely used in clinical practice to make treatment decisions for AML. We evaluated the efficacy of interphase FISH to detect chromosome anomalies in the workup of young (<60 years) patients with AML.Methods: Study subjects were enrolled in E1900, a front-line Eastern Cooperative Oncology Group (ECOG) clinical trial for AML. This is an on-going Phase III clinical trial with Daunorubicin dose-intensification ± Gemtuzumab-Ozogamicin consolidation therapy prior to stem cell transplant. This trial opened December 2002; as of February 2005, 223 patients were enrolled. The protocol was designed to collect bone marrow for both cytogenetic and FISH studies at study entry (diagnosis). Cytogenetic studies were done by local laboratories with results reviewed centrally by the ECOG Cytogenetics Committee. Each case was classified as acceptable or unacceptable based on predefined ECOG cytogenetic criteria. FISH for each patient was performed in the ECOG FISH laboratory at Mayo Clinic and utilized eight probe sets to detect t(8;21), t(9;22), t(11;var), t(15;17), inv(16), +8, −5/5q, and −7/7q (Vysis, Downer Grove, IL).Results: 64 (29%) of 223 specimens had incomplete cytogenetic and/or FISH results. We analyzed the remaining 159 (71%) specimens with complete cytogenetic and FISH results. Results for each specimen were classified by probe set into one of the following categories:Normal cytogenetics and normal FISH;Abnormal cytogenetics and abnormal FISH for the anomaly the probe was designed to detect;Abnormal cytogenetics and abnormal FISH for an anomaly the probe was not primarily designed to detect;Normal cytogenetics and abnormal FISH;Abnormal cytogenetics and normal FISH; orAbnormal cytogenetics and abnormal FISH that further defined the karyotype. [Display omitted] The concordance rate between cytogenetic and FISH results ranged from 97 to 100% for all probe sets and kappa analysis for concordance had a p value of <0.0001. Of the total 159 cases, discrepancies between FISH and cytogenetic results occurred in only 4 cases; two with normal cytogenetics and abnormal FISH and two with abnormal cytogenetics and normal FISH results.Conclusions: The high level of agreement between cytogenetics and FISH demonstrates the accuracy of a panel of 8 FISH probe sets for the detection of significant abnormalities in AML. The data from this investigation support the use of FISH as an adjunct in cases of failed cytogenetic analyses to increase the yield of useful cytogenetic results in large cooperative trials. Furthermore, because of the strong correlation between cytogenetics and FISH, our results demonstrate the potential of FISH as a follow-up study of minimal residual disease in ECOG trials.

BiRD (Biaxin®/Revlimid®/Dexamethasone) Combination Therapy (Rx) Results in High Complete Remissions (CR) and Overall Responses in Myeloma (MM) with Poor Prognostic Features.

Revlimid® (R) or lenalidomide is the leading clinical compound in a new group of drugs called IMiD®s, which have immunomodulatory properties. In contrast with thalidomide (T) R has significantly less neurological toxicity. Several phase I and II trials with R showed promising results in relapsed/refractory MM. Our group demonstrated that Bi augments tumor mass reduction and improves responses in patients (pts) receiving low-dose T and/or D. We report the initial results of a phase II trial exploring the combination of Bi plus R plus D (BiRD) in newly diagnosed MM.Methods: The trial is designed to accrue 35 pts. Thus far 28 pts have been accrued between Nov. 2004 and Aug. 2005 of which 22 pts (13 male and 9 female) are eligible for evaluation. R is given orally (po) at a dose of 25 mg daily on days 1–21 of a 28-day cycle. D is given po at a dose of 40 mg once weekly. Bi is given po at 500 mg twice daily. All pts also receive low dose aspirin (ASA)(81mg) once daily as thrombosis prophylaxis, prophylactic sulfamethoxazole/trimethoprim, and a proton pump inhibitor. Responses are defined according to the EBMTR. In addition, the category of near-CR (nCR) is defined as a CR with a positive immunofixation. A minimum of 2 cycles is required for response assessment; all pts are assessed on an intent-to-treat basis.Results: Pt characteristics are as follows: median age of 62 years (range 36–77), hemoglobin of 11.4g/dL (range 7.2–15.1), platelets of 247k/uL (range 51–526), β2m of 4.0mg/L (range 0.8–5.1), CRP of 1.0mg/dL (range 0.3–8.4), creatinine of 1.3mg/dL (range 0.5–3.1), albumin of 3.6g/dL (range 2.3–4.9), and calcium of 8.9mg/dL (range 6.9–11.2). Conventional cytogenetics was normal in all pts; FISH abnormalities are as follows: trisomy 11 (2 pts), tetrasomy 11 (1 pt), del13q14(5 pts), t(4,14)(1pt), t(11,14)(2 pts). 52% of the pts are stage IIIa, 13% are stage IIIb, 35% are stage IIa. Responses are summarized in the table below. Of the 22 evaluable pts, 21(95%) have achieved an objective response (>PR) within 1–2 months of Rx with the remaining pt continues to respond. Nearly one third of pts have achieved either a CR(6/22) or a nCR(1/22-continuing on Rx). The remaining 14 pts(63%) achieved a PR. Of those pts who achieved a PR, 6/14 pts(43%) had >90% reduction in the initial paraprotein, while 12/14 pts(86%) had >75% decrement. 5 pts are now off study (3 pts in CR going to PBSCT and 2 due to toxicities). So far 15 pts have experienced grade ≥3 adverse events. These include, anemia(4.5%), neutropenia(4.5%), thrombocytopenia(4.5%), increased liver enzymes(4.5%), anxiety(4.5%), insomnia(9%), tremors(4.5%), hyperglycemia(4.5%), syncope(4.5%), Stevens Johnson's(4.5%) and colonic perforation(4.5%). DVT occurred in 3(13.6%) patients, all off ASA, of which 2 (9%) had an associated PE, one of whom died. No grade 4 toxicities have been otherwise observed. Three pts have undergone successful stem cell harvest of which one has undergone transplantation with melphalan 200. Engraftment was successful by day 12.Conclusions: BiRD is highly effective and safe in the initial Rx of MM. Stem cell harvest is not impaired after BiRD induction. Supported in part by the LLS SCOR grant and K23CA109260-01Response Evaluation# of Pts%Total22100Overall Response2195CR627.3n-CR14.5PR1463.6SD14.5

FCR-3 as Frontline Therapy for Patients with Chronic Lymphocytic Leukemia (CLL).

The combination regimen of Fludarabine, Cyclophosphamide, and Rituximab (FCR) has produced high overall response (OR) rates and complete remission (CR) rates in patients receiving this as initial treatment for CLL. Earlier studies have shown that dose-dense or dose-intensified regimens of single-agent Rituximab produced higher response rates in patients with CLL. The current regimen, FCR-3, was based on the hypothesis that increasing the amount of Rituximab in the combination might improve response rates and remission duration. Doses of chemotherapy were Fludarabine 25 mg/m2/Dx3, and Cyclophosphamide 250 mg/m2/Dx3 given monthly. Rituximab was given at 375 mg/m2 as the first dose and 500 mg/m2 for all subsequent doses. On each day of the chemotherapy a dose of Rituximab was given so 3 doses were given monthly. With the previous FCR regimen 45% of patients achieved a CR or nPR and had <5% CD19+ 5 positive cells in the marrow at the completion of 3 cycles of therapy (rapid response, RR). The median time to progression (TTP) in that group has not been reached; it was 3 years in patients who did not achieve that endpoint (p<.001). The current protocol aimed to increase that response rate from 45% to 60%. Sixty-five patients were treated. Eighty percent were men. The median age was 59 years (27–82). Rai Stage 3–4 disease was present in 25% of patients. Median WBC count was 92.4 x 103/ul (7.9–363). Median B2-microglobulin was 3.8 (1.6–10.1). Unfavorable FISH abnormalities were present in 35% of 52 evaluable patients. Somatic hypermutation status was available for 44 patients; 61% were unmutated. ZAP-70 expression analysis performed by immunostaining or flow cytometry was positive in 62% of 47 evaluable patients. Results of FCR-3 in comparison to FCR are shown in the table.3 Cycles6 CyclesNo.RR(%)ORCRFlow<5%FCR30045957282FCR-36545946574PCR negativity using consensus primer PCR was achieved in 49% of patients at the end of therapy. Median number of days between courses ranges from 29–35 per course (overall range 27–95). Eighty-five percent of patients completed 6 cycles. No patient has progressed with a median follow-up of 10 months. With limited follow-up the addition of 3 doses of Rituximab to FC chemotherapy does not appear to provide greater benefit than one dose.

Low Level Amplification (Duplication) of 1q21 in Myeloma and Prognosis; the Role of CKS1B.

Introduction: Molecular cytogenetic studies have revealed that, to a great extent, the heterogeneity of myeloma is largely dictated by the underlying genetic and cytogenetic aberrations present in the clonal plasma cells. Most recently the group from the University of Arkansas identified strong prognostic associations with an increased level of gene expression of a cell cycle associated gene, CKS1B. CKS1B favors cell cycle progression by promoting degradation of p27 with release of the cyclin dependent kinases and entry into mitosis.Patients and methods: To further test this hypothesis we studied: a) via FISH for CKS1B amplification in a cohort of patients treated at the Mayo Clinic with high dose chemotherapy and stem cell support, as well as a group of patients with cytogenetically defined hypodiploidy, and b) a cohort of myeloma patients that were studied by gene expression profiling. Gene expression analysis was performed on CD138-enriched plasma-cell RNA using Affymetrix U133A chips (Affymetrix, Santa Clara, CA).Results: Of 159 patients studied 46 exhibited FISH abnormalities consistent with increase number of signals for CKS1B (30%): amplification was marginal or low in 44 cases, and in only two cases the ratio between CKS1B and control probe was greater than 2.0. Therefore, the predominant pattern is one of gene duplication rather than amplification where gene copy usually exceeds 10 and ratio with control probe exceeds 5 (e.g. HER-2/neu amplification). Patients with CKS1B duplication had a higher prevalence of chromosome 13 deletion (72%), and t(4;14) (29%). The presence of CKS1B duplication was more frequent (53%) among 19 patients with hypodiploidy. Using gene expression data for CKS1B, we found a positive association with t(14;16)(q32;q23). The level of CKS1B gene expression positively correlated with the PCLI (p<0.0001) but there was heterogeneity in this relationship (r2 = 0.34). We found no correlation between the expression level of CKS1B and p27 (r2 = 0.008, p = 0.37). While CKS1B gene duplication had a negative effect on survival this effect was weak (29.9 versus 38 months, p = 0.124. Median follow-up is 55 months) and disappeared when the variable was entered into the multivariate model including PCLI, B2-microglobulin and all the major genetic abnormalities by FISH. Likewise the level of expression of CKS1B determined by gene expression only carried prognostic significance when extreme levels of expression were utilized as a prognostic variable.Discussion: In this study we show that increase copy number of CKS1B is present in one third of patients with MM (majority of these being gene duplication) and seem to be associated with a shorter overall survival, but the net effect of this is rather weak in our series. Further study is needed to understand its potential role as a progression factor in the plasma cell neoplasms.

Thyroid hormone deficiency in abnormal larvae of the Japanese flounder Paralichthys olivaceus

In large-scale rearing of juveniles of the Japanese flounder Paralichthys olivaceus, a certain morphological abnormality occurred spontaneously in 4% of the fish. These fish showed a slight but clearly different appearance from any developmental stage of this species, and did not settle when all the other juveniles in the same tank completed metamorphosis and had settled. From comparisons of external and internal structures between the normal and the abnormal fish, the abnormality was attributed to unbalanced progress of metamorphosis, mainly due to metamorphic stasis. The thyroid of the abnormal fish was apparently activated morphologically. In addition, serum thyroxine (T4) concentrations in the abnormal fish were reduced to less than 1/10 of that of normal fish. After 14 days of T4 treatment (0.1 p.p.m) of the abnormal fish, all the abnormal characteristics disappeared, and the fish recovered to normal, suggesting normal responsiveness to thyroid hormones in peripheral tissues, whereas thiourea treatment (30 p.p.m., 14 days) further delayed metamorphosis. These results suggest that these abnormal fish were suffering from thyroid hormone deficiency, and were unable to secrete a sufficient amount of thyroid hormone to complete metamorphosis.

Myxobolus buri, the myxosporean parasite causing scoliosis of yellowtail, is synonymous with Myxobolus acanthogobii infecting the brain of the yellowfin goby

: Myxobolus buri Egusa, 1985, is a well-documented myxosporean parasite that causes the scoliosis of cultured yellowtail Seriola quinqueradiata. A similar parasite has been described as Myxobolus acanthogobii Hoshina, 1952, from the brain of the yellowfin goby Acanthogobius flavimanus, although this parasite is not associated with skeletal abnormalities in host fish. The present study aimed to re-examine the identification of these two parasites by morphological and molecular analyses. Morphological characteristics (e.g. presence of the intercapsular processes and several folds on the sutural ridges) and spore dimensions were not distinguishable between the two species and were consistent with those in the original descriptions. Molecular analysis indicated that small subunit rRNA gene sequences shared 100% identity between the two parasites. Consequently, it can be concluded that M. buri is synonymous with M. acanthogobii, and thus this parasite can be reassigned as M. acanthogobii.

Increased incidence of chromosomal abnormalities in embryos from polycystic ovarian syndrome patients

ObjectiveIncreased rates of first trimester pregnancy losses have been reported in polycystic ovarian syndrome (PCOS) patients. Increased PAI-1 levels, hyperinsulinaemia-driven, homocysteinaemia and increased body weight have been described as contributors to the high miscarriage rates in these women. To study the incidence of chromosomal abnormalities on day 3 embryos from PCOS patients included in a preimplantational genetic diagnosis (PGD) programme. To check if the selection of euploid embryos for transfer can diminish pregnancy loss rates in these patients.DesignRetrospective study from January 2000 to March 2004.Materials and methodsPGD was performed in 12 PCOS patients because of previous implantation failures or recurrent miscarriages (n= 87 embryos). Couples with sperm account <5 millions spermatozoa/ml, abnormal Fish spermatozoa and =38 years of age were excluded. Pregnancy, implantation and miscarriage rates were compared with a control group of 18 pgd cycles for sex linked diseases carried out during the same period (<38 years). χ2 test was used for statistical comparisons.ResultsAn increased rate of abnormal embryos were found (* P<0.001). When euploid embryos for 13, 15, 16,28,21,22,X and Y chromosomes were transferred , all pregnancies had a successful outcome.Conclusion ObjectiveIncreased rates of first trimester pregnancy losses have been reported in polycystic ovarian syndrome (PCOS) patients. Increased PAI-1 levels, hyperinsulinaemia-driven, homocysteinaemia and increased body weight have been described as contributors to the high miscarriage rates in these women. To study the incidence of chromosomal abnormalities on day 3 embryos from PCOS patients included in a preimplantational genetic diagnosis (PGD) programme. To check if the selection of euploid embryos for transfer can diminish pregnancy loss rates in these patients. Increased rates of first trimester pregnancy losses have been reported in polycystic ovarian syndrome (PCOS) patients. Increased PAI-1 levels, hyperinsulinaemia-driven, homocysteinaemia and increased body weight have been described as contributors to the high miscarriage rates in these women. To study the incidence of chromosomal abnormalities on day 3 embryos from PCOS patients included in a preimplantational genetic diagnosis (PGD) programme. To check if the selection of euploid embryos for transfer can diminish pregnancy loss rates in these patients. DesignRetrospective study from January 2000 to March 2004. Retrospective study from January 2000 to March 2004. Materials and methodsPGD was performed in 12 PCOS patients because of previous implantation failures or recurrent miscarriages (n= 87 embryos). Couples with sperm account <5 millions spermatozoa/ml, abnormal Fish spermatozoa and =38 years of age were excluded. Pregnancy, implantation and miscarriage rates were compared with a control group of 18 pgd cycles for sex linked diseases carried out during the same period (<38 years). χ2 test was used for statistical comparisons. PGD was performed in 12 PCOS patients because of previous implantation failures or recurrent miscarriages (n= 87 embryos). Couples with sperm account <5 millions spermatozoa/ml, abnormal Fish spermatozoa and =38 years of age were excluded. Pregnancy, implantation and miscarriage rates were compared with a control group of 18 pgd cycles for sex linked diseases carried out during the same period (<38 years). χ2 test was used for statistical comparisons. ResultsAn increased rate of abnormal embryos were found (* P<0.001). When euploid embryos for 13, 15, 16,28,21,22,X and Y chromosomes were transferred , all pregnancies had a successful outcome. An increased rate of abnormal embryos were found (* P<0.001). When euploid embryos for 13, 15, 16,28,21,22,X and Y chromosomes were transferred , all pregnancies had a successful outcome. Conclusion

A latex agglutination test for the field determination of abnormal vitellogenin productionin male fishes contaminated by estrogen mimics

Estrogen mimics are pollutants present in the aquatic environment. These compoundsinduce abnormalities in the reproductive system of male fishes, which lead to a total orpartial male feminization, or to their demasculinization. Ultimately, these alterations couldlead to a disappearance of the total contaminated fish population. Moreover, these toxicsubstances possess the capacity to mimic endogenous estrogens and to induce the abnormalproduction of vitellogenin (VTG) in male and immature fishes. The purpose of thisresearch was to develop an easy, specific, cheap and fast method for diagnosing thecontamination of male fishes by estrogen mimics, using VTG as biomarker. The selectedmethod is based on a reverse latex agglutination test (rLAT), developed with monoclonalantibodies specific of this biomarker. The development of this VTG-rLAT has involved,firstly, the purification of carp VTG to produce monoclonal antibodies, specifics of thisprotein. One of these antibodies was selected to recover latex particles (diameter:1 µm). Finally,the immunoreactivity of the VTG-rLAT was verified with different fish plasma samples from males treatedwith 17β-estradiol and non-treated males or females in vitellogenesis.

Selecting appropriate animal models and experimental designs for endocrine disruptor research and testing studies.

Evidence that chemicals in the environment may cause developmental and reproductive abnormalities in fish and wildlife by disrupting normal endocrine functions has increased concern about potential adverse human health effects from such chemicals. US laws have now been enacted that require the US Environmental Protection Agency (EPA) to develop and validate a screening program to identify chemicals in food and water with potential endocrine-disrupting activity. EPA subsequently proposed an Endocrine Disruptor Screening Program that uses in vitro and in vivo test systems to identify chemicals that may adversely affect humans and ecologically important animal species. However, the endocrine system can be readily modulated by many experimental factors, including diet and the genetic background of the selected animal strain or stock. It is therefore desirable to minimize or avoid factors that cause or contribute to experimental variation in endocrine disruptor research and testing studies. Standard laboratory animal diets contain high and variable levels of phytoestrogens, which can modulate physiologic and behavioral responses similar to both endogenous estrogen as well as exogenous estrogenic chemicals. Other studies have determined that some commonly used outbred mice and rats are less responsive to estrogenic substances than certain inbred mouse and rat strains for various estrogen-sensitive endpoints. It is therefore critical to select appropriate biological models and diets for endocrine disruptor studies that provide optimal sensitivity and specificity to accomplish the research or testing objectives. An introduction is provided to 11 other papers in this issue that review these and other important laboratory animal experimental design considerations in greater detail, and that review laboratory animal and in vitro models currently being used or evaluated for endocrine disruptor research and testing. Selection of appropriate animal models and experimental design parameters for endocrine disruptor research and testing will minimize confounding experimental variables, increase the likelihood of replicable experimental results, and contribute to more reliable and relevant test systems.

Occurrence and Effects of Endocrine Disrupting Chemicals in the Environment

This review focuses on environmental chemicals that mimic the activity of the endogenous estrogen, 17b-estradiol ~E2!, and their effects on wildlife, because it is the most studied area of endocrine disruption. The potential human health impact of endocrine disruptors is beyond the scope of this paper and readers should refer to other publications as reported by Crisp et al. in 1998 and Safe in 1995. The concentrations of endocrine disrupting chemicals in effluents from sewage treatment plants ~STPs! are summarized and compared. By utilizing in vitro and in vivo assays, environmental estrogens from STPs and other point source pollutants have been shown to induce reproductive abnormalities in fish and wildlife. Though treatment systems of sewage facilities can reduce the amounts of endocrine disrupting chemicals, the removal efficiency can still be improved to further reduce estrogenic effects upon aquatic organisms. Up to date, a lack of comprehensive research impedes the ability to correlate estrogenic chemicals to community- and ecosystem-level effects. Thus, total ecosystem evaluations such as species diversity and impact on keystone species and understudied nonpoint source pollution should be two primary focuses of future research.

Occurrence and removal of plant sterols in pulp and paper mill effluents

Pulp and paper mill effluents (PPMEs) are believed to contain some naturally occurring endocrine disrupting chemicals (EDCs) such as phytosterols or plant sterols, which may be related to a variety of physiological and morphological abnormalities in fish inhabiting receiving waters. Analyses of effluent streams from two Canadian pulp mills confirmed the presence of five different phytosterols in PPMEs. Sterol contents of biologically treated as well as untreated effluents are presented. ββ-Sitosterol, ββ-sitostanol (or stigmastanol), and campesterol formed the major fraction of plant sterols in PPMEs, accounting for 70 % or more of the total phytosterol content measured. Sterols entering the activated sludge treatment systems were generally removed from the effluents with average removal efficiencies of 72 and 66% for the two mills surveyed. Sorption and biodegradation may be responsible for the observed removal of plant sterols across the biological wastewater treatment systems. Key words: ββ-sitosterol, stigmastanol, campesterol, removal and mass flow of phytosterols or plant sterols, hormone disruptors, pulp mill effluents, activated sludge, industrial wastewater treatment.

Susceptibility to abnormal (kinetotic) swimming fish correlates with inner ear carbonic anhydrase-reactivity

Larval cichlid fish ( Oreochromis mossambicus) were kept at hypergravity (hg; centrifuge) for 6 h. Following the transfer to 1 g (i.e. stopping the centrifuge), animals were separated into normally and abnormally (kinetotic) swimming individuals (the latter were swimming kinetotically, i.e. performing spinning movements). Subsequently, carbonic anhydrase- (CA-) reactivity was histochemically demonstrated and densitometrically determined in inner ear maculae. It was found that both the total macular CA-reactivity as well as the difference in reactivates between left and right maculae were significantly lower in normally swimming hg-animals as compared to the kinetotically behaving hg-fish ( P<0.0001). This result is in complete agreement with closely related studies carried out on the calcium incorporation of inner ear otoliths and indicates that a regulatory mechanism, which adjusts otolithic calcium carbonate incorporation towards the gravity vector, acts via activation/deactivation of macular CA.

Exploring polycythaemia vera with fluorescence in situ hybridization: additional cryptic 9p is the most frequent abnormality detected.

Between 1986 and 2001, 220 patients with polycythaemia vera (PV) were studied using conventional cytogenetics. Of 204 evaluable patients, 52 (25.4%) had clonal abnormalities. The recurrent chromosomal rearrangements were those of chromosome 9 (21.1%), del(20q) (19.2%), trisomy 8 (19.2%), rearrangements of 13q (13.4%), abnormalities of 1q (11.5%), and of chromosomes 5 and 7 (9.6%). Subsequent analysis of 32 patients, performed at follow-up of up to 14.8 years, revealed new clonal abnormalities in five patients and the disappearance of an abnormal clone in four. Eleven patients remained normal up to 11.5 years and seven patients maintained an abnormality for over 10 years. Fifty-three patients were studied retrospectively using interphase fluorescence in situ hybridization (I-FISH), utilizing probes for centromere enumeration of chromosomes 8 and 9, and for 13q14 and 20q12 loci. Conventional cytogenetics demonstrated clonal chromosome abnormalities in 23% of these 53 patients. The addition of I-FISH increased the detection of abnormalities to 29% and permitted clarification of chromosome 9 rearrangements in an additional 5.6% of patients. FISH uncovered rearrangements of chromosome 9 in 53% of patients with an abnormal FISH pattern, which represented the most frequent genomic alteration in this series.

A comparative study on morphological differences in the olfactory system of red sea bream ( Pagrus major) and black sea bream ( Acanthopagrus schlegeli) from wild and cultured stocks

The improving technology in artificial propagation of aquatic animals is having a direct effect on increasing aquaculture fishery production of many aquatic food species throughout the world. High-valued fish species are being produced en masse and released into the wild to augment populations depleted due to over-fishing and habitat degradation. This study was conducted to determine the effect of procedural conditions in mariculture hatcheries on the olfactory systems of fish in Kagoshima Prefecture, Japan, and to compare the results with fish from wild stocks. Red sea bream ( Pagrus major) and black sea bream ( Acanthopagrus schlegeli) were suitable candidates for this study because of their economic importance and history in mariculture production, especially ongoing restocking of natural populations via stock enhancement programs in Japan. An extensive survey revealed a high percentage of fish with an abnormal single olfactory nasal opening. The single abnormal nasal opening contrasted strongly with a pair of natural nasal openings, an anterior and a posterior nares, in normal fish of both species whereby olfactory water is sampled when the fish swims. Ultrastructural observation of lamellar topography demonstrated not only a substantial variation in sensory epithelial distribution patterns in red sea bream of cultured stock but also showed that the sensory epithelial area in abnormal cultured fish was significantly smaller ( P<0.005) than the lamellar area in wild fish. The densities/mm 2 of microvillous and ciliated olfactory receptor neurons in both species were not significantly different when normal fish were compared with abnormal fish. Nonetheless, there were some notable irregularities in distribution patterns of the two receptor neurons on the lamellar surface. In abnormal red sea bream, the density of ciliated nonsensory cells was relatively higher than the normal fish. The kinocilia in cultured fish (normal red sea bream with anterior and posterior nares; CNR, 6.45±0.83 and abnormal red sea bream with a single nasal opening; CAR, 8.05±1.39 μm SD) were significantly longer ( P<0.05) than the ones observed in wild fish (5.46±1.39 μm SD). Further, ciliary beat cycle and metachrony were evident in normal fish olfactory epithelia but were usually not evident in abnormal fish. The results indicated that gross and micromorphological abnormalities in the olfactory systems of fish are a consequence of certain procedural condition(s) in mariculture facilities. The physiological and behavioral implications of morphological deformities in fish olfactory systems are discussed, especially in the context of ensuring production of healthy, normal fish for marine ranching programs and ecological preservation.

Regularities in Primary Production, Secchi Depth and Fish Yield and a New System to Define Trophic and Humic State Indices for Lake Ecosystems

General relationships between phytoplankton production, chlorophyll, total, dissolved and particulate phosphorus, Secchi depth, humic level, trophic level, fish production and latitude are described by regression equations using an extensive “Soviet” data base covering a wide domain of lake characteristics and a European data base. New systems for defining lake trophic and humic status are presented. The results may be used for more precise estimates of fundamental lake properties and for many practical issues of lake management, e.g., predictions of fish catch. We have used strict chlorophyll-a concentrations for every trophic class and we have omitted Secchi depth from the trophic classes, since Secchi depth and other variables strongly related to water clarity (like suspended particulate matter and particulate organic carbon) depend on autochthonous production, allochthonous influences and resuspension. We have used the Secchi depth as a simple operational measure of the effective depth of the photic zone. It has also been shown that among these lakes there exist a very strong relationship between primary production and latitude. In fact, 74% of the variability among the lakes in mean summer primary production can be statistically related to variations in latitude. These data also show a strong relationship between primary production and fish yield, which can be used to address many fundamental issues in lake management, like “normal and abnormal fish production”.

Long-term increased bioaccumulation of mercury in largemouth bass follows reduction of waterborne selenium

Average mercury concentrations in largemouth bass from Rogers Quarry in east Tennessee were found to increase steadily following the elimination of selenium-rich discharges of fly ash to the quarry in 1989. From 1990 to 1998, mean mercury concentrations (adjusted to compensate for the covariance between individual fish weight and mercury concentration) in bass rose from 0.02 to 0.61 mg/kg. There was no indication that the rate increase was slowing or that mercury concentrations in fish were approaching a plateau or steady state. Mean selenium concentrations in bass declined from 3 to 1 mg/kg over the first five years of the study, but remained at 1–1.5 mg/kg (about twice typical concentrations in bass from local reference sites) for the last three years of the study. Gross physical abnormalities were common in fish from the site in the first three years after elimination of fly ash discharges but disappeared after two more years. Although it remains possible that other chemical or physical changes related to fly ash disposal in the system were associated with increased mercury bioaccumulation, the most likely explanation is that selenium played a critical role. It appears as though aqueous selenium enrichment was capable of having a profound effect on mercury bioaccumulation in this system but at the cost of causing a high incidence of gross abnormalities in fish. However, it is possible that selenium concentrations between the national ambient water quality criterion for the protection of aquatic life, 5 μg/l, and that now found in Rogers Quarry (<2 μg/l) could reduce mercury bioaccumulation without causing adverse effects on aquatic biota and fish-eating wildlife.

Growth, feed utilization and body lipid content of gilthead seabream ( Sparus aurata) fed increasing lipid levels and fish meals of different quality

The present study shows the effects of feeding gilthead seabream, 70 to 400 g, with nine diets containing three different lipid levels (15, 22 and 28% on a dry weight basis), combined with two types of fish meal of different quality. In addition, feed processing effects (extruded vs. pelletized) were also compared for the diet containing 22% lipid. The diets prepared with the high quality fish meal promoted better growth than those prepared with the low quality one. Protein utilization was enhanced by a sparing effect of dietary lipid at 22%. This lipid level needed to be increased up to 28% with low quality meal to effectively spare dietary protein. A significant increase in fish growth with higher dietary lipid levels, in both high and low quality fish meal diets, was only related to a significant increase in total body lipid content in high quality fish meal diets, and an excess of lipid may have been the cause of hepatocyte abnormalities in fish fed the highest lipid levels. The effect of pellet processing was apparent in low quality fish meal diets, where a significantly better growth performance was obtained in fish fed the extruded diet. There were no significant differences among all treatments in feed intake, and values were similar to those suggested by commercial fish feed producers, for similar fish size and water temperatures. Values for feed conversion ratio (FCR) were lower than 1.6 for all diets.

Biomarkers Indicate Health Problems in Brown Bullheads from the Industrialized Schuylkill River, Philadelphia

We used biomarkers to assess the health of two populations of brown bullheads Ameiurus nebulosus one from an urban, industrialized section of the Schuylkill River (SR), Philadelphia, Pennsylvania, and the other from Hopkins Pond (HP), in suburban New Jersey, which was not affected by industry. We evaluated health using histopathology, condition factor (K), hepatosomal index (HSI), prevalence of parasites, health assessment index (HAI), and population age structure. Sediment analysis revealed that both sites contained similar levels of polynuclear aromatic hydrocarbons (PAHs). Brown bullheads from the SR were longer and heavier than brown bullheads from HP and exhibited more external lesions. There were no hepatic lesions in brown bullheads from either site. Condition factor was higher in SR fish, but due to length differences between the two populations, was not a reliable indicator of general health. Hepatosomal index was similar between both populations, although the presence of numerous cestodes in the visceral organs, including the liver, of HP brown bullheads confounded the interpretation of the HSI. The incidence of parasitism was significantly greater in HP fish than in SR fish. The HAI was significantly greater in SR brown bullheads, indicating more organ abnormalities in SR fish than in HP fish, including more abnormalities of the fins, barbels, and mouth. Population age structure was older for HP brown bullheads than for SR fish, indicating a shorter lifetime for SR brown bullheads. Although PAH levels were similar between the two sites, SR brown bullheads were less healthy than HP brown bullheads, as indicated by several biomarkers. Histopathology, health assessment index, and population age structure were the most informative biomarkers.

Fluorescence In Situ Hybridization to Assess Aneuploidy for Chromosomes 7 and 8 in Hematologic Disorders

Stored, fixed cell suspensions of bone marrows from 70 patients karyotyped over a three-year period for myelodysplastic syndrome (MDS) or related hematologic conditions were retrospectively studied in two series using centromeric probes for chromosomes 7 and 8. Series I consisted of patient samples with numerical and/or structural abnormalities of chromosomes 7 or 8, matched with chromosomally normal samples from about the same time period. Series II consisted of consecutive MDS patient samples as well as patient samples in which one or more cells had numerical or structural abnormalities of 7 and 8. In both series, probes for chromosomes 7 and 8 were applied in each case and at least 100 nuclei were scored for each probe for the distribution of one, two, or three signals. Twenty-seven cases had clonal abnormalities by routine cytogenetics (RC): 12 with monosomy 7; one with monosomy 8; five with trisomy 8; nine with clonal abnormalities other than 7 or 8 aneuploidy. Eleven cytogenetically normal cases gave abnormal interphase FISH (IF) results; one was subsequently confirmed by metaphase FISH analysis to have a clonal structural abnormality of chromosome 7; one case with a trisomy 8 clone, in remission by RC, showed 35% of cells by IF with three signals for chromosome 8; one case had heteromorphic chromosomes by FISH. Of eight remaining cases, five (four with −7 and one with +8 by IF) were among 22 cases of cytogenetically normal MDS. Three remaining cases (two with +8 and one with both +7 and +8 by IF) had AML or MPD. The high rate of possible undetected monosomy 7, among MDS cases in particular, suggests all MDS cases should be screened by IF.