Impact of historical legacy pesticides on achieving legislative goals in Europe.

Pesticides are widely used in agriculture to optimise food production. However, the movement of pesticides into water bodies negatively impacts aquatic environments. The European Union (EU) aims to make food systems fair, healthy and environmentally friendly through its current Farm to Fork strategy. As part of this strategy, the EU plans to reduce the overall use and risk of chemical pesticides by 50 % by 2030. The attainment of this target may be compromised by the prevalence of legacy pesticides arising from historical applications to land, which can persist in the environment for several decades. The current EU Farm to Fork policy overlooks the potential challenges of legacy pesticides and requirements for their remediation. In this review, the current knowledge regarding pesticide use in Europe, as well as pathways of pesticide movement to waterways, are investigated. The issues of legacy pesticides, including exceedances, are examined, and existing and emerging methods of pesticide remediation, particularly of legacy pesticides, are discussed. The fact that some legacy pesticides can be detected in water samples, more than twenty-five years after they were prohibited, highlights the need for improved EU strategies and policies aimed at targeting legacy pesticides in order to meet future targets.

Transcriptomic response and immunological responses to chimpanzee adenovirus- and MVA viral-vectored vaccines for RSV in healthy adults.

Cohorts of healthy younger adults (18-50yrs) and healthy older adults (60-75yrs) were immunized intramuscularly or intranasally with an adenovirus-vectored RSV vaccine (PanAd3-RSV) as a prime dose and boosted with PanAd3-RSV or a poxvirus-vectored vaccine (MVA-RSV) encoding the same insert. Whole blood gene expression was measured at baseline, 3- and 7-days post vaccination. Intramuscular prime vaccination with PanAd3-RSV induced differential expression of 643 genes (DEGs, FDR < 0.05). Intranasal prime vaccination with PanAd3-RSV did not induce any differentially expressed genes (DEGs) in blood samples at 3 days post vaccination. Intranasally primed participants showed greater numbers of DEGS on boosting than intramuscularly primed participants. The most highly enriched biological processes related to DEGs after both prime and boost vaccination were type-1 interferon related pathways, lymphocytic and humoral immune responses.

A risk ranking of pesticides in Irish drinking water considering chronic health effects.

This paper presents a novel scoring system which facilitates a relative ranking of pesticide risk to human health arising from contaminated drinking water. This method was developed to identify risky pesticides to better inform monitoring programmes and risk assessments. Potential risk was assessed considering pesticide use, chronic human health effects and environmental fate. Site-specific soil conditions, such as soil erodibility, hydrologic group, soil depth, clay, sand, silt, and organic carbon content of soil, were incorporated to demonstrate how pesticide fate can be influenced by the areas in which they are used. The indices of quantity of use, consequence and likelihood of exposure, hazard score and quantity-weighted hazard score were used to describe the level of concern that should be attributed to a pesticide. Metabolite toxicity and persistence were also considered in a separate scoring to highlight the contribution metabolites make to overall pesticide risk. This study presents two sets of results for 63 pesticides in an Irish case study, (1) risk scores calculated for the parent compounds only and (2) a combined pesticide-metabolite risk score. In both cases the results are assessed for two locations with differing soil and hydrological properties. The method developed in this paper can be adapted by pesticide users to assess and compare pesticide risk at site level using pesticide hazard scores. Farm advisors, water quality monitors, and catchment managers can apply this method to screen pesticides for human health risk at a regional or national level.

Cyclotron production of Ac-225 for targeted alpha therapy.

The feasibility of producing Ac-225 by proton irradiation of Ra-226 in a cyclotron through the reaction Ra-226(p,2n)Ac-225 has been experimentally demonstrated for the first time. Proton energies were varied from 8.8 to 24.8 MeV and cross-sections were determined by radiochemical analysis of reaction yields. Maximum yields were reached at incident proton energies of 16.8 MeV. Radiochemical separation of Ac-225 from the irradiated target yielded a product suitable for targeted alpha therapy of cancer.

Long-term (7 to 8-year) experience with finasteride in men with benign prostatic hyperplasia.

OBJECTIVES: To evaluate the effects of finasteride, a specific type II 5-alpha-reductase inhibitor, on symptoms of benign prostatic hyperplasia, prostate volume, and urinary flow during a 7 to 8-year period. METHODS: A total of 190 men with symptomatic benign prostatic hyperplasia and enlarged prostates entered one of two Phase II double-blind 3 to 6-month studies. Of these, 156 patients continued taking open-label finasteride, and more than 70 patients completed 7 to 8 years of treatment. The symptoms were scored using a patient self-administered modified Boyarsky symptom questionnaire. Prostate volume was measured by magnetic resonance imaging or ultrasonography, and the maximal urinary flow rate was assessed noninvasively. RESULTS: Treatment with finasteride for 7 to 8 years led to sustained improvement in symptoms, reduction in prostate volume (28% from baseline), and increased urinary flow (median 2.5 mL/s from baseline). Decreases in dihydrotestosterone (86%) and prostate-specific antigen (54%) levels were also maintained. Long-term finasteride treatment was safe and generally well tolerated. CONCLUSIONS: Long-term treatment with finasteride was well tolerated and resulted in durable symptom relief and improvement in prostate volume and urinary flow.

Mechanics of the antebrachial interosseous membrane: response to shearing forces.

The interosseous membrane functions to maintain the structural integrity of the forearm; however, the age-related mechanical properties of the intact membrane are incompletely defined. The purpose of this study is to quantify the mechanical properties of the interosseous membrane by applying a quasi-equilibrium shearing force between the radius and ulna. A 5-cm section of the central (main and accessory bundles) fiber from 11 (5 male and 6 female) fresh-frozen cadaver arms (age, 74 +/- 6.9 years) was isolated with the radial and ulnar attachments, measured, and positioned into the testing apparatus. A shear force was applied at 12.7 mm/min to produce a quasi-equilibrium state, and the applied force and elongation were monitored. The average ultimate force, ultimate strength, and elastic modulus were 1101 +/- 191 N, 13.98 +/- 4.85 MPa, and 135.29 +/- 41.57 MPa, respectively. Age-related changes were also noted with an increase in thickness by 0.056 mm per year of age and a decrease in fiber stiffness of 4.78 MPa/yr. The age-related mechanical properties of the intact interosseous membrane are necessary for modeling forearm stability in normal and pathologic conditions. This information is required for analysis of repair or reconstructive procedures.

Molecular analysis of estrogen induction of preproenkephalin gene expression and its modulation by thyroid hormones.

Estrogen receptors (ER) and thyroid hormone receptors (TR) are ligand-dependent nuclear transcription factors. Estrogen-induced preproenkephalin (PPE) gene expression in the hypothalamus is directly related to estrogen-induced lordosis behavior in the rat. In the present study, we showed that the PPE mRNA level in the ventromedial hypothalamus of female rats was significantly decreased by ovariectomy. This decrease was reversed by estrogen replacement in a dose- and time-dependent manner. Using transient transfection and electrophoretic mobility shift assays (EMSA), functional estrogen response elements (ERE) were identified between -437 and -145 base pairs (bp) of the rat PPE gene promoter region. Two ERE-like elements are present between -405 and -364 of the rat PPE gene promoter, which bind ERalpha as demonstrated by EMSA. Estrogen produced a dose-dependent increase in CAT activity in cotransfection assays with ERalpha expression vector and a 437PPE-CAT reporter construct containing 437 bp of the rat PPE gene promoter and the CAT reporter gene. This estrogen-induced PPE promoter activity was inhibited by liganded-TR in transient cotransfection assays. Analysis of DNA-protein interactions by EMSA revealed that both ERalpha and TR (alpha1 and beta1) could bind to the EREs in the rat PPE gene promoter. Furthermore, estrogen induction of PPE mRNA in the ventromedial hypothalamus of the ovariectomized female rat was significantly attenuated by concomitant administration of triiodothyronine. These results suggest that estrogen regulation of the hypothalamic PPE gene expression is mediated through an estrogen-receptor complex directly interacting with the functional EREs in its promoter region; and that this estrogen effect can be modified by thyroid hormones.

Dopexamine hydrochloride does not modify hemodynamic response or tissue oxygenation or gut permeability during abdominal aortic surgery.

PURPOSE: To assess the effects of intraoperative infusion of dopexamine (a DA-1 and B2 adrenoreceptor agonist) on hemodynamic function, tissue oxygen delivery and consumption, splanchnic perfusion and gut permeability following aortic cross- clamp and release. METHODS: In a randomised double blind controlled trial 24 patients scheduled for elective infrarenal abdominal aortic aneurysm repair were studied in two centres and were assigned to one of two treatment groups. Group I received a dopexamine infusion starting at 0.5 microg x kg(-1) x min(-1) increased to 2 microg x kg(-1) x min(-1) maintaining a stable heart rate; Group II received a placebo infusion titrated in the same volumes following induction of anesthesia. Measured and derived hemodynamic data, tissue oxygen delivery and extraction and gut permeability were recorded at set time points throughout the procedure. RESULTS: Dopexamine infusion (0.5 -2 microg x kg x min(-1)) was associated with enhanced hemodynamic function (MAP 65 +/- 5.5 vs 92 +/- 5.7 mm Hg, P = <0.05) only during the period of aortic cross clamping. However, during the most part of infrarenal abdominal aortic surgery, dopexamine did not reduce systemic vascular resistance index, mean arterial pressure nor oxygen extraction compared with the control group. The lactulose/ rhamnose permeation ratio was elevated above normal in both groups (0.22 and 0.29 in groups I and II respectively). CONCLUSIONS: Dopexamine infusion (0.5 -2 microg x kg(-1) x min(-1)) did not enhance hemodynamic function and tissue oxygenation values during elective infrarenal abdominal aortic aneurysm repair.

Design and initial evaluation of a low-cost 3-Tesla research system for combined optical and functional MR imaging with interventional capability.

A 3-Tesla research system has been developed for functional and interventional magnetic resonance imaging (MRI) procedures on animal models based on a low field niche spectrometer. Use of two stages of fourth harmonic frequency multiplication has allowed us to produce a high-frequency spectrometer with good frequency stability based on a low-frequency direct digital synthesizer. The system has been designed with the ability to introduce interventional tools such as biopsy needles, radiofrequency (RF) electrodes, and fiber optics for optical spectroscopy and thermal ablation as well as drug infusions to allow function to be studied in the presence of external challenges. Full MR-compatible physiologic support capability allows animals to be maintained in a stable condition over extended periods of study. Functional MR images have been acquired by using gradient echoes (TR/TE = 40/12 msec) from the rat whisker barrel cortex using electrical stimulation (5-V, 1.5-mA, 1-msec pulses at 5 Hz via two needle electrodes inserted into the rat whisker pad). Initial results using respiratory gas challenges of 100% N(2), 100% O(2), and 10% CO(2) have shown excellent agreement between single wavelength (633 nm) optical and functional MR time series with subsecond time resolution. The 1-mm copper electrodes for interventional radiofrequency ablation procedures were easily visualized in the superior colliculus by using gradient echo sequences. This novel, low-cost, high field system appears to be a useful research tool for functional and interventional studies of rat brain and allows concurrent optical spectroscopy. J. Magn. Reson. Imaging 2001;13:87-92.

Effect of the aromatase inhibitor vorozole on estrogen and progesterone receptor content of rat mammary carcinomas induced by 1-methyl-1-nitrosourea.

Vorozole, a nonsteroidal aromatase inhibitor, impedes the post-initiation stage of chemically induced mammary carcinogenesis. While various aspects of vorozole's effects on mammary carcinoma development have been investigated, little attention has been directed to determining the estrogen receptor (ER) and progesterone receptor (PR) content of mammary carcinomas that arise despite vorozole treatment. Female Sprague-Dawley rats were given an i.p. injection of 50mg MNU/kg body weight at 21 days of age and placed on diet supplemented with 0 or 3 mg vorozole/kg, which had no effect on mammary tumor development. Histologically confirmed carcinomas were evaluated for ER and PR by immunohistochemistry. In the control group, 78.8% of carcinomas were ER positive with an ER content ranging from 13.8 to 40.0%, similar to ER content of mammary ductal epithelial cells from non-carcinogen treated animals. PR content ranged from 4.4 to 45.2% and also was similar to levels of PR observed in ductal epithelial cells. ER was not correlated with PR in mammary carcinomas (r = 0.05, p > 0.80), whereas there was a significant correlation in ductal epithelium (r = 0.86, p = 0.006). In vorozole-treated rats, no ER negative carcinomas were observed and overall ER expression by vorozole was elevated (p < 0.03). All carcinomas from vorozole-treated rats expressed PR (2.5-60.2%) and correlation between ER and PR content was numerically greater in carcinomas from vorozole-treated animals (r = 0.42, p = 0.09). These data, which are considered hypothesis generating, provide evidence that low doses of vorozole in the diet select for mammary carcinomas with an increased ER positive phenotype.

A comparison of the histopathology of premalignant and malignant mammary gland lesions induced in sexually immature rats with those occurring in the human.

The injection of sexually immature female rats with 1-methyl-1-nitrosourea results in a rapid induction of premalignant and malignant mammary gland lesions within 35 days of carcinogen administration. This model affords the opportunity for investigators to study the process of mammary carcinogenesis over a very short latency and to investigate early events in this process. We have recently published on various aspects of this system including the histology of the lesions induced, the time frame of their occurrence, and their dependence on ovarian hormones for their maintenance and growth. In this report we present evidence that many aspects of the histopathology of mammary lesions in this model system are similar to those occurring in humans. We also discuss aspects of the human disease, which are not recapitulated in this model.

Increased 5alpha-reductase and normal 11beta-hydroxysteroid dehydrogenase metabolism of C19 and C21 steroids in a young population with polycystic ovarian syndrome.

OBJECTIVE: To test the hypothesis that 5alpha-reductase (5alphaR) and 11beta-hydroxysteroid dehydrogenase (11beta-HSD) activity are increased in adolescent and young-adult women with PCOS and that an altered regulation of the hypothalamic-pituitary-adrenal (HPA) axis occurred in these subjects. DESIGN: Prospective non-randomized study in an academic research environment. PATIENTS: Eleven women, aged 14 to 25 years, were studied who were at least one year post-menarche and who had a diagnosis of PCOS based on a history of oligomenorrhea and elevated total and or free serum testosterone. INTERVENTION: 24-Hour urinary metabolites were assessed in nine subjects and five underwent stimulation with ovine corticotropin releasing factor (oCRF). OUTCOME MEASURES: C19 and C21 steroid urinary metabolite 5-alpha/5-beta pairs, 11-oxo/11-hydroxy products and the ratio of the total 5-alpha/5-beta reduced and 11-oxo/11-hydroxy products were compared to values in control women. Urinary cortisol (F) (sum of conjugated and free, and free F) and total F metabolites (the sum of THE, THF, 5alpha-THF, cortolones, and cortols) were determined. A 1 microg/kg oCRF stimulation test was performed with timed samples determined for plasma ACTH and serum F levels. RESULT: The 24-hour total and free urinary F were not different from control. However, the total F metabolites were markedly elevated (7922+/-2666 vs 5418+/-1549 microg/24 h, p<0.01). A marked increase in the total 5-alpha reduced C19 and C21 metabolites was observed in the PCOS population vs control (5084+/-1977 vs 2681+/-1188 microg/24 h, p<0.01). The total urinary 11-oxo/11-hydroxy metabolite ratio was not different, p=0.23. The basal values and response of both ACTH and F to oCRF stimulation were not different from those of controls. CONCLUSION: There is a marked increase in 5alphaR metabolism of both C19 and C21 steroids in younger women with PCOS.

Effect of fixation and epitope retrieval on BrdU indices in mammary carcinomas.

Studies in which 5-bromo-2'-deoxyuridine (BrdU) is used to quantify rates of cell proliferation are conducted prospectively. Therefore, the opportunity exists to select conditions that optimize detection of the BrdU epitope. The objective of this study was to quantify the extent to which the BrdU epitope was masked by formalin vs methacarn fixation in the assessment of cell proliferation. Mammary carcinomas from animals pulse-labeled with BrdU were trisected. A portion was frozen and the remaining two portions were fixed in 10% neutral buffered formalin or methacarn for 24 hr, processed, embedded in paraffin, and sections stained for incorporated BrdU using a peroxidase immunohistochemical staining technique. Antigen retrieval techniques also were applied to formalin-fixed sections. Fixation in methacarn gave the highest labeling index (16.4%), which was comparable to that observed in unfixed frozen sections (17.5%). Formalin fixation alone dramatically suppressed the labeling index (0.3%), which was only partially recovered using various antigen retrieval techniques (2.1-8.1%). Methacarn fixation is recommended for prospective studies in which BrdU detection is planned because of the quantitative recovery of epitope and the simplicity of the approach.

Classification of premalignant and malignant lesions developing in the rat mammary gland after injection of sexually immature rats with 1-methyl-1-nitrosourea.

Premalignant and malignant stages of mammary carcinogenesis can be rapidly induced by injecting female rats i.p. with 1-methyl-1-nitrosourea (MNU)3 at 21 days of age. In this paper, the characteristics of this model are briefly reviewed and the histology of the lesions induced is presented and compared to those that occur in humans. Malignant mammary lesions induced in rats injected with MNU at 21 days of age are compared with the lesions that develop when MNU is administered to 50-day-old female rats.

Cisapride does not alter gastric volume or pH in patients undergoing ambulatory surgery.

PURPOSE: To evaluate the efficacy of 20 mg cisapride p.o. in reducing residual gastric volume and pH in adult ambulatory surgical patients. METHODS: Using a prospective randomised double-blind controlled design, we administered either 20 mg cisapride p.o. or placebo preoperatively to 64 ASA 1-2 ambulatory surgical patients. Following induction of anesthesia we measured volume and pH of residual gastric contents, using blind aspiration through an orogastric tube. Parametric data were analysed using unpaired, one tail Students' t test. Non-parametric data were analysed using Fishers Exact test and Chi square analysis. Statistical significance was accepted at the probability level of < 0.05. RESULTS: Residual gastric volumes were similar in the two groups (19.5 +/- 23.8, 23.9 +/- 24.4 ml), in the cisapride and placebo groups respectively, P=0.24). Data shown are mean (+/- SD). The proportions of patients with a residual gastric volume exceeding 0.4 ml x kg(-1) were similar in the two groups (4 of 28, and 8 of 23 patients in the cisapride and placebo groups respectively, P=0.09). The pH of the residual gastric contents were similar in the cisapride and placebo groups (1.6 +/- 0.5, 1.4 +/- 0.5, respectively, P=0.26). The proportions of patients with pH < 2.5 was also similar in the cisapride and placebo groups (21 of 25, and 20 of 21 patients respectively, P=0.2). CONCLUSIONS: Preoperative administration of 20 mg cisapride p.o. to patients scheduled for outpatient surgery does not alter either the volume or the pH of gastric contents. Its use in this setting is of no apparent clinical benefit.

Conjugated linoleic acid-enriched butter fat alters mammary gland morphogenesis and reduces cancer risk in rats.

Conjugated linoleic acid (CLA) is a potent cancer preventive agent in animal models. To date, all of the in vivo work with CLA has been done with a commercial free fatty acid preparation containing a mixture of c9,t11-, t10,c12- and c11,t13-isomers, although CLA in food is predominantly (80-90%) the c9,t11-isomer present in triacylglycerols. The objective of this study was to determine whether a high CLA butter fat has biological activities similar to those of the mixture of free fatty acid CLA isomers. The following four different endpoints were evaluated in rat mammary gland: 1) digitized image analysis of epithelial mass in mammary whole mount; 2) terminal end bud (TEB) density; 3) proliferative activity of TEB cells as determined by proliferating cell nuclear antigen immunohistochemistry; and 4) mammary cancer prevention bioassay in the methylnitrosourea model. It should be noted that TEB cells are the target cells for mammary chemical carcinogenesis. Feeding butter fat CLA to rats during the time of pubescent mammary gland development reduced mammary epithelial mass by 22%, decreased the size of the TEB population by 30%, suppressed the proliferation of TEB cells by 30% and inhibited mammary tumor yield by 53% (P < 0.05). Furthermore, all of the above variables responded with the same magnitude of change to both butter fat CLA and the mixture of CLA isomers at the level of CLA (0.8%) present in the diet. Interestingly, there appeared to be some selectivity in the uptake or incorporation of c9,t11-CLA over t10,c12-CLA in the tissues of rats given the mixture of CLA isomers. Rats consuming the CLA-enriched butter fat also consistently accumulated more total CLA in the mammary gland and other tissues (four- to sixfold increases) compared with those consuming free fatty acid CLA (threefold increases) at the same dietary level of intake. We hypothesize that the availability of vaccenic acid (t11-18:1) in butter fat may serve as the precursor for the endogenous synthesis of CLA via the Delta9-desaturase reaction. Further studies will be conducted to investigate other attributes of this novel dairy product.

A novel mutation of the human luteinizing hormone receptor in 46XY and 46XX sisters.

We report a novel homozygous mutation of the LH receptor (LHR) gene in three siblings: two 46XY and one 46XX. The 46XY siblings presented with female external genitalia, primary amenorrhea, and lack of breast development. Hormonal evaluation revealed a markedly elevated LH level with a low testosterone level, which failed to increase after human CG stimulation. Enzymatic deficiencies of testosterone biosynthesis were eliminated as possible etiologies. Histologic analysis of the inguinal gonads in a 46XY sibling revealed no Leydig cells; Sertoli cells, spermatogonia, and primary spermatocytes were seen. The 46XX sibling had female external genitalia, normal breast development, and primary amenorrhea. Hormonal analyses showed markedly elevated LH levels and low plasma 17 beta-estradiol levels. Genetic analysis of the LHR revealed a homozygous missense mutation at exon 11 of the LHR gene. Guanine was replaced by adenine (GAA-->AAA), resulting in a substitution of lysine for glutamic acid (glu) at amino acid position 354 of the receptor. This mutation is located in the extracellular domain adjacent to the first transmembrane helix of the LHR. Glutamic acid at position 354 of the LHR has been highly conserved throughout evolution. Functional analysis of the LHR mutation, using an in vitro mutagenesis-transfection assay, demonstrated complete loss of function, indicated by the lack of cAMP production after human CG stimulation in transfected human embryonic kidney 293 cells. Screening of family members demonstrated heterozygosity for the mutation, indicating autosomal recessive inheritance. Delineation of the specific genetic defect in this family confirms recent reports that a single mutation in the LHR gene causes male pseudohermaphroditism in 46XY subjects and primary amenorrhea in 46XX subjects. More importantly, it also defines a new region of the LHR molecule that is critical for biologic activity.

Ovarian hormone dependence of pre-malignant and malignant mammary gland lesions induced in pre-pubertal rats by 1-methyl-1-nitrosourea.

The experiments reported in this study were designed to examine the question of whether a mammary epithelial cell's independence from hormonal requirements is established at the time of carcinogenic initiation, or whether the emergence of hormone independence is associated with the process of tumor progression. A newly developed rat model of mammary carcinogenesis was used in which the latency period to lesion detection is very short and in which the frequencies of both pre-malignant and malignant mammary lesions can be quantified. Two experiments were conducted in Sprague-Dawley rats injected with 50 mg MNU/kg body wt at 21 days of age. In the first experiment 47 animals were ovariectomized after the detection of a mammary tumor of palpable size. Forty-six of the 47 tumors assessed, all of which were subsequently classified as mammary gland adenocarcinomas, regressed to <50% of their initial volume within 14 days of bilateral ovariectomy. However, both pre-malignant and malignant mammary gland lesions were observed when animals were killed. In Experiment 2 a total of 60 rats were ovariectomized 7 days after MNU was injected. At 35 days post carcinogen ovariectomized animals had a higher incidence of intraductal proliferations than sham-operated controls (P = 0.03); there was no effect of ovariectomy on the incidence of ductal carcinoma in situ or carcinoma. The multiplicity of intraductal proliferations was increased by 58% in ovariectomized rats (P = 0.12), but the number of mammary carcinoma per rat was reduced (3.8 vs. 1.57, P = 0.02). These data are consistent with the hypotheses that the progression of pre-malignant to malignant lesions is inhibited in the mammary gland by ovariectomy and that the hormone independent phenotype can be conferred at the time of carcinogenic initiation.

The identification of 5 alpha-reductase-2 and 17 beta-hydroxysteroid dehydrogenase-3 gene defects in male pseudohermaphrodites from a Turkish kindred.

Male pseudohermaphroditism (MPH) is characterized by incomplete differentiation of male genitalia in the presence of testicular tissue. Enzymatic defects involving androgen synthesis or action are causes of MPH. We studied the molecular genetics of a large isolated inbred Turkish kindred with MPH due to either 5 alpha-reductase-2 (SRD5A2) or 17 beta-hydroxysteroid dehydrogenase-3 (17 beta HSD3) gene defects. Using single strand DNA conformational polymorphism analysis and DNA sequencing, a new mutation in exon 5 of SRD5A2 gene was detected in certain male pseudohermaphrodites from this kindred. This single base deletion (adenine) resulted in a frame shift at amino acid position 251 resulting in the addition of 23 amino acids at the carboxyl-terminal of this 254-amino acid isozyme. Transfection expression of the mutant isozyme in CV1 cells showed a complete loss of enzymatic activity in the conversion of [14C]testosterone to dihydrotestosterone, without a change in the messenger ribonucleic acid level compared to that of the wild-type isozyme. Analysis of the 17 beta HSD3 gene in other male pseudohermaphrodites from this kindred revealed a single point mutation (G-->A) at the boundary between intron 8 and exon 9, disrupting the splice acceptor site of exon 9. In this kindred, in addition to the identification of male pseudohermaphrodites with either a homozygous SRD5A2 or 17 beta HSD3 gene defect, other male pseudohermaphrodites were found to be genetically more complex: e.g. homozygous for the SRD5A2 defect and heterozygous for the 17 beta HSD3 defect, or homozygous for the 17 beta HSD3 defect and heterozygous for the SRD5A2 defect. Also, phenotypically normal carriers were identified with either one or both gene defects. Homozygous male pseudohermaphrodites with SRD5A2 or 17 beta HSD3 gene defects were phenotypically distinguishable by the presence of mild gynecomastia in the latter. Hormone data were consistent with the particular homozygous gene defect. In summary, we show 1) the novel existence of two gene defects, SRD5A2 and 17 beta HSD3, each causing MPH within a large isolated Turkish kindred; 2) that the two defects segregate independently and may be inherited from two different progenitors; and 3) analysis of a new mutation in exon 5 of SRD5A2 gene, supporting the functional importance of the carboxyl-terminal of 5 alpha-reductase-2 isozyme.