A case report of neurological adverse events caused by short-term and low-dose treatment of mitotane: The role of therapeutic drug monitoring.

RATIONALE: Low-dose mitotane has been widely used for many decades in patients with advanced adrenocortical carcinoma (ACC), which exhibited good safety profiles compared with the high-dose regimen. The clinical efficacy and toxicity of mitotane are closely related to its plasma concentration, and therapeutic drug monitoring (TDM) is recommended. Until now, no severe adverse drug reaction (ADR) related to the toxic plasma level after a short-term treatment of low-dose mitotane has been published. PATIENT CONCERNS: A 50-year-old Chinese female presented with severe neurological adverse events related to a toxic plasma levels of 42.8 mg/L after 4 months treatment of low-dose mitotane. DIAGNOSES: During the course of therapy, no other medication could cause neurological adverse events. Therefore, we suspected a high sensitivity to the side effect of mitotane related to a toxic plasma level. INTERVENTIONS: Treatment of mitotane was stopped. OUTCOMES: The trough plasma concentration of mitotane decreased to 18.7 mg/mL after one and a half months, and the neurological symptoms gradually improved after drug discontinuance. LESSONS: The present case provides the first report of severe neurological adverse events induced by the short-term use of low-dose mitotane for adjuvant treatment in a patient with ACC, indicating that potentially severe ADR can also occur when using low-dose regimen in the early stage of treatment. TDM and early recognition could result in a favorable outcome.

Exquisite sensitivity of adrenocortical carcinomas to induction of ferroptosis.

Adrenocortical carcinomas (ACCs) are rare and highly malignant cancers associated with poor survival of patients. Currently, mitotane, a nonspecific derivative of the pesticide DDT (1,1-(dichlorobiphenyl)-2,2-dichloroethane), is used as the standard treatment, but its mechanism of action in ACCs remains elusive. Here we demonstrate that the human ACC NCI-H295R cell line is remarkably sensitive to induction of ferroptosis, while mitotane does not induce this iron-dependent mode of regulated necrosis. Supplementation with insulin, transferrin, and selenium (ITS) is commonly used to keep NCI-H295R cells in cell culture. We show that this supplementation prevents spontaneous ferroptosis, especially when it contains polyunsaturated fatty acids (PUFAs), such as linoleic acid. Inhibitors of apoptosis (zVAD, emricasan) do not prevent the mitotane-induced cell death but morphologically prevent membrane blebbing. The expression of glutathione peroxidase 4 (GPX4) in H295R cells, however, is significantly higher when compared to HT1080 fibrosarcoma cells, suggesting a role for ferroptosis. Direct inhibition of GPX4 in H295R cells led to high necrotic populations compared to control, while cotreatment with ferrostatin-1 (Fer-1) completely reverted ferroptosis. Interestingly, the analysis of public databases revealed that several key players of the ferroptosis pathway are hypermethylated and/or mutated in human ACCs. Finally, we also detected that growth hormone-releasing hormone (GHRH) antagonists, such as MIA602, kill H295R cells in a nonapoptotic manner. In summary, we found elevated expression of GPX4 and higher sensitivity to ferroptosis in ACCs. We hypothesize that instead of treatment with mitotane, human adrenocortical carcinomas may be much more sensitive to induction of ferroptosis.

Foetal exposure to mitotane/Op'DDD: Post-natal study of four children.

OBJECTIVE: Mitotane/Op'DDD is used in the treatment of adrenocortical carcinoma and for other causes of hypercortisolism. Mitotane inhibits cortisol secretion and displays adrenolytic and antitumor actions. This compound is a metabolite of the pesticide and endocrine disruptor DDT (dichlorodiphenyltrichloroethane) and is classified among teratogenic compounds worldwide. However, little is known about its effects on human development. DESIGN: The outcome of four children exposed to mitotane during their intrauterine life was examined. PATIENTS: Patients having conceived while taking mitotane, or with detectable mitotane plasma levels, were retrospectively recruited via the French COMETE and FIRENDO networks. MEASUREMENTS: Mitotane in maternal plasma, adrenocortical hormones in children. RESULTS: Three women treated with mitotane gave birth to four children. During early pregnancy, all patients had detectable mitotane plasma levels (0.9, 2.4 and 6.7 mg/L, respectively). During pregnancy, no foetal malformations were detected. The four exposed newborns presented at birth with apparently normal adrenal function and genitalia. One twin female had a low birthweight. Evaluation at birth and after 3 months, 2 years and 7 years of follow-up showed no significant neurological abnormality. Evaluation of adrenocortical functions showed no cortisol deficiency. CONCLUSIONS: Unexpectedly, exposure of these four children to mitotane during foetal life seemed to have no clear teratogenic effect. However, considering the sub-therapeutic mitotane concentrations used here, the small number of cases, and because long-term follow-up is unknown, we strongly advise not to take mitotane during pregnancy and still recommend avoiding pregnancy, at least as long as mitotane plasma levels remain detectable.

The adrenal specific toxicant mitotane directly interacts with lipid membranes and alters membrane properties depending on lipid composition.

Mitotane (o,p'.-DDD) is an orphan drug approved for the treatment of adrenocortical carcinoma. The mechanisms, which are responsible for this activity of the drug, are not completely understood. It can be hypothesized that an impact of mitotane is mediated by the interaction with cellular membranes. However, an interaction of mitotane with (lipid) membranes has not yet been investigated in detail. Here, we characterized the interaction of mitotane and its main metabolite o,p'-dichlorodiphenyldichloroacetic acid (o,p'-DDA) with lipid membranes by applying a variety of biophysical approaches of nuclear magnetic resonance, electron spin resonance, and fluorescence spectroscopy. We found that mitotane and o,p'-DDA bind to lipid membranes by inserting into the lipid-water interface of the bilayer. Mitotane but not o,p'-DDA directly causes a disturbance of bilayer structure leading to an increased permeability of the membrane for polar molecules. Mitotane induced alterations of the membrane integrity required the presence of phosphatidylethanolamine and/or cholesterol. Collectively, our data for the first time characterize the impact of mitotane on the lipid membrane structure and dynamics, which may contribute to a better understanding of specific mitotane effects and side effects.

Inhibitory effects of mitotane on viability and secretory activity in mouse gonadotroph cell lines.

Mitotane represents the mainstay medical treatment for metastatic, inoperable or recurrent adrenocortical carcinoma. Besides the well-known adverse events, mitotane therapy is associated also with endocrinological effects, including sexual and reproductive dysfunction. The majority of male patients undergoing adjuvant mitotane therapy show a picture of hypogonadism, characterized by low free testosterone and high sex hormone binding globulin levels and unmodified LH concentrations. Since mitotane has been shown to have direct pituitary effects, we investigated whether mitotane may influence both cell viability and function of gonadotroph cells in the settings of two pituitary cell lines. We found that mitotane reduces cell viability, induces apoptosis, modifies cell cycle phase distribution and secretion of gonadotroph cells. The present data strengthen previous evidence showing a direct mitotane effect at pituitary level and represent a possible explanation of the lack of LH increase following decrease in free testosterone in patients undergoing adjuvant mitotane therapy.

Ecotoxicological risk assessment linked to the discharge by hospitals of bio-accumulative pharmaceuticals into aquatic media: The case of mitotane.

The release of hospital wastewater into the urban sewer networks contributes to the general contamination of aquatic media by pharmaceutical residues. These residues include bio-accumulative pharmaceuticals that lead to increased risk for ecosystems because they can concentrate in organisms and food chains, and therefore reach toxic levels. In order to assess the ecotoxicological risks linked to this particular category of residues, we have developed a specific method, by combining a theoretical calculation of pollutant concentrations in organisms to estimate Body Residue (BR), and ecotoxicity biomarkers in fish cell lines, enabling the calculation of a Critical Body Residue (CBR). This method finally results in the calculation of a specific risk quotient (Qb=BR/CBR), characterizing the risk linked to this type of pollutant. This method was applied to mitotane, a bio-accumulative pharmaceutical typically found in hospital wastewater, in the framework of an exposure scenario corresponding to the discharge of all the hospital wastewaters into the Rhone River which flows through the city of Lyon, France. This approach leads to risk quotients (Qb and Qbg) much higher than those found with the classical approach, i.e. Q=PEC/PNEC (Predictive Environmental Concentration/Predictive Non Effect Concentration)=0.0006. This difference in the appreciation of risk is important when using cytotoxicity as the criterion for measuring the toxicity of mitotane (Qb=0.056) and it is even greater when the criterion used is genotoxicity (Qbg=6.8). This study must be now consolidated by taking the biomagnification of the pharmaceuticals into consideration.

The effect of mitotane on viability, steroidogenesis and gene expression in NCI­H295R adrenocortical cells.

Mitotane, also known as o,p'­DDD or (RS)­1­chl-oro­2­[2,2­dichloro­1­(4­chlorophenyl)­ethyl]­benzene, is an adrenal cortex-specific cytotoxic drug used in the therapy of adrenocortical carcinoma (ACC). The drug also inhibits steroidogenesis, however, the mechanisms of its anticancer and antisteroidogenic effects remain unknown. At present, data on the impact of mitotane on cell viability and the regulation of genes encoding proteins associated with steroids synthesis in the adrenal cortex, including cortisol and dehydroepiandrosterone sulfate (DHEAS), are limited and contradictory. In the present study, the effect of 24­h mitotane treatment on viability of the ACC cell line, NCI­H295R, was analyzed, identifying a decrease in cell viability and an increase in caspase­3 and ­7 activities. Mitotane treatment also led to decreased cortisol and DHEAS concentration in the culture media. Concomitantly, mitotane resulted in decreased mRNA levels of two cytochromes P450 (CYP11A1 and CYP17A1), mRNAs encoding proteins involved in the synthesis of cortisol and DHEAS. Mitotane did not affect mRNA levels of cyclin dependent kinase inhibitor 1A (encoding p21) and MYC (encoding cMyc). cMyc and p21 are key transcription factors associated with cell cycle regulation. However, mitotane inhibited expression of transforming growth factor ß1 gene, encoding a potent inhibitor of cell proliferation and steroidogenesis. PRKAR1A, a protein kinase A regulatory subunit, is involved in the activation of steroidogenesis. PRKAR1A mRNA levels were reduced following 24­h treatment with mitotane. Results indicate that mitotane markedly inhibited expression of genes involved in steroidogenesis, secretion of cortisol and DHEAS. Reduced expression of TGFB1 cannot account fully for the effect of mitotane on CYP11A1 and CYP17A1. We hypothesized that reduced viability of NCI­H295R cells in the presence of mitotane may be a result of apoptosis triggered by increased caspase­3 and ­7 activities. Since p21 and cMyc mRNA levels were stable in the presence of mitotane, the mechanism by which caspase­3 and ­7 are induced remains unknown.

Organochlorine pesticides and polychlorinated biphenyls in sediments and fish from freshwater cultured fish ponds in different agricultural contexts in north-eastern France.

Organochlorine pesticides (HCB, HCH with α-, ß-, and γ isomers, heptachlor, cis-heptachlor epoxyde, trans-heptachlor epoxyde, endosulfan with α- and ß isomers, sulfate endosulfan, o,p'-DDT, p,p'-DDT, o,p'-DDE, p,p'-DDE, o,p'-DDD, p,p'-DDD, chlorothalonil, alachlor, aldrin, dieldrin, methoxychlor, oxychlordane, chlordane with α- and γ isomers, p,p'-dicofol and o,p'-dicofol) and indicators PCBs (IUPAC nos. 28, 52, 101, 118, 138, 153, and 180) were studied both in sediments and muscles of farmed fish species (Cyprinus carpio and Perca fluviatilis). Samples were collected from fish ponds located in the hydrographic basin of the Moselle River (Lorraine Region, France). OCPs and PCBs were present at low concentrations both in sediments and fish muscles. Concerning sediments, ∑DDTs revealed concentrations ranging from 0.2 to 2.30 ng g(-1) dw and ∑PCBs ranged from 0.3 to 3.5 ng g(-1) dw. Concerning fish muscles, the highest concentrations in OCPs were those of p,p'-DDE, with average concentrations of 0.57±0.44 ng g(-1) ww for carp and 0.58±0.29 ng g(-1) ww for perch. The contamination profiles proved to be different depending on the fish species. Indeed, HCH-isomers, HCB, and dieldrin were detected only for the carp and always at low concentrations. For example, the highest concentration of HCHs was observed for ß-HCH with a mean value of 0.64±0.15 ng g(-1) ww for carp. As for PCBs, the levels of ∑PCBs ranged from 0.3 to 6.4 ng g(-1) ww in carp muscles and from 0.90 to 5.60 ng g(-1) ww in perch muscles.

Comparative CYP-dependent binding of the adrenocortical toxicants 3-methylsulfonyl-DDE and o,p'-DDD in Y-1 adrenal cells.

The environmental pollutant 3-MeSO(2)-DDE [2-(3-methylsulfonyl-4-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethene] is an adrenocortical toxicant in mice, specifically in the glucocorticoid-producing zona fasciculata, due to a cytochrome P450 11B1 (CYP11B1)-catalysed bioactivation and formation of covalently bound protein adducts. o,p'-DDD [2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane] is toxic and inhibits steroidogenesis in the human adrenal cortex after bioactivation by unidentified CYPs, but does not exert any toxic effects on the mouse adrenal. As a step towards determining in vitro/in vivo relationships for the CYP-catalysed binding and toxicity of 3-MeSO(2)-DDE and o,p'-DDD, we have investigated the irreversible protein binding of these two toxicants in the murine adrenocortical cell line Y-1. The irreversible binding of 3-MeSO(2)-DDE previously demonstrated in vivo was successfully reproduced and could be inhibited by the CYP-inhibitors etomidate, ketoconazole and metyrapone. Surprisingly, o,p'-DDD reached similar levels of binding as 3-MeSO(2)-DDE. The binding of o,p'-DDD was sensitive to etomidate and ketoconazole, but not to metyrapone. Moreover, GSH depletion increased the binding of 3-MeSO(2)-DDE, but not of o,p'-DDD, indicating an important role of GSH conjugation in the detoxification of the 3-MeSO(2)-DDE-derived reactive metabolite. In addition, the specificity of CYP11B1 in activating 3-MeSO(2)-DDE was investigated using structurally analogous compounds. None of the analogues produced histopathological lesions in the mouse adrenal in vivo following a single i.p. injection of 100 mg/kg body weight, but two of the compounds were able to decrease the irreversible binding of 3-MeSO(2)-DDE to Y-1 cells. These results indicate that the bioactivation of 3-MeSO(2)-DDE by CYP11B1 is highly structure-dependent. In conclusion, both 3-MeSO(2)-DDE and o,p'-DDD bind irreversibly to Y-1 cells despite differences in binding and adrenotoxicity in mice in vivo. This reveals a notable in vitro/in vivo discrepancy, the contributing factors of which remain unexplained. We consider the Y-1 cell line as appropriate for studies of the cellular mechanisms behind the adrenocortical toxicity of these substances.

DDT- and DDE-induced disruption of ovarian steroidogenesis in prepubertal porcine ovarian follicles: a possible interaction with the main steroidogenic enzymes and estrogen receptor beta.

We evaluated impact of DDT isomers, o, p'- DDT [1, 1-dichloro-2, 2-bis (p, p'-chlorophenyl) ethylene] and p, p'-DDT [1, 1, 1-trichloro-2, 2-bis (p-chlorophenyl) ethane], and their metabolites, o, p'-DDE and p, p'-DDE, on ovarian steroidogenesis. All these compounds, except for p, p'-DDT, demonstrated estrogenic effects on steroid secretion in co-cultures of porcine prepubertal granulosa and theca cells. p,p'-DDT decreased progesterone and estradiol release, which was reversed by the addition of testosterone. In contrast, o, p'-DDT inhibited progesterone secretion with parallel stimulation of basal and testosterone-stimulated estradiol release. DDEs stimulated progesterone and estradiol secretion. The fluorometric assay confirmed that p,p'-DDE, o,p'-DDT, and o,p'-DDE stimulated aromatase activity. Western blots indicated that o,p-DDT and o,p'-DDE diminished the expression of estrogen receptor beta (ERbeta). This study demonstrated the isomer-dependent action of DDT in pig ovarian cells. We propose that DDT could disrupt ovarian steroidogenesis either by interfering with main steroidogenic enzymes or affecting ERbeta.

Effect of single and repeated in vitro exposure of ovarian follicles to o,p'-DDT and p,p'-DDT and their metabolites.

The aim of the presented study was to compare the effect of o,p'-DDT [1,1-dichloro-2,2-bis-(p,p'-chlorophenyl)-ethylene] and p,p'-DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)-ethane] and their metabolites DDE and DDD on estradiol secretion by ovarian follicles, the target organs of environmental estrogens. Theca interna (Tc) and granulosa cells (Gc) were collected from medium size porcine follicles and cultured as a monolayer. The cells were initially cultured for 24 h to allow attachment to the plates and then media were changed for the new ones and o,p'-DDT and p,p'-DDT and their metabolites: o,p'-DDE, p,p'-DDE and o,p'-DDD were added at doses of 4, 40, 400 ng and 4 microg/ml medium to investigate dose-dependent effects. Media were collected after 24 h and frozen for estradiol content determination. When the effect of single and repeated exposure was investigated, the lowest dose of 4 ng/ml and the highest one of 4 microg/ml were chosen on the basis of the results of Experiment 1. o,p'-DDT exerted antiestrogenic action at all doses used while its metabolites and p,p'-DDT and its metabolites decreased estradiol secretion only when present in the medium at a dose of 4 ng/ml. The highest doses caused the increase in estradiol secretion. Parent o,p'-DDT and its metabolites showed antiestrogenic action after single exposure to 4 ng/ml while parent p,p'-DDT and its metabolites caused estrogenic action. All investigated compounds, except o,p'-DDT, increased estradiol secretion after single exposure to the dose of 4 microg/ml. Repeated exposure resulted in a massive antiestrogenic action of all investigated chemicals. In conclusion, our study points to time-dependent effect of DDT and its metabolites on ovarian follicles with the strongest estrogenic properties observed after single exposure and antiestrogenic action caused by repeated exposure. Given the duration of folliculogenesis, one can imagine many different potential mechanisms by which DDT could influence steroidogenesis.

Short-term exposure of Chinook salmon (Oncoryhnchus tshawytscha) to o,p-DDE or DMSO during early life-history stages causes long-term humoral immunosuppression.

We evaluated the effect of short-term exposures to a xenobiotic chemical during early life-history stages on the long-term immune competence of chinook salmon (Oncoryhnchus tshawytscha). Immersion of chinook salmon eggs in a nominal concentration of o,p-dichlorodiphenyldichloroethylene (o,p-DDE; 10 ppm) for 1 hr at fertilization followed by immersion in the same dose for 2 hr at hatch resulted in a significant reduction in the ability of splenic leukocytes from fish 1 year after treatment to undergo blastogenesis upon in vitro stimulation with lipopolysaccharide. We also observed that the vehicle, dimethyl sulfoxide (DMSO), caused a significant reduction in the ability of the splenic leukocytes to express surface immunoglobin M (SIgM) at this time. The concentration of o,p-DDE in a pooled sample of whole fry from this treatment was 0.53 microg/g lipid 1 month after first feeding but was undetectable in all other treatments. Mortality rate, time to hatch, fish length, and weight were unaffected by treatment with o,p-DDE. Similarly, sex ratios, gonadal development, and concentrations of plasma estradiol and 11-ketotestosterone were not affected by the treatment. In addition, we found no evidence that plasma lysozyme concentrations or the mitogenic responses of splenic leukocytes to concanavalin A or polyinosinic-polycytidylic acid were influenced by the treatment. In this experiment, a brief period of exposure to o,p-DDE or DMSO during early development was able to induce long-term effects on humoral immune competence of chinook salmon. Such immunosuppression may increase susceptibility to disease, which may in turn be critical to regulating the population.

Toxicity of o,p'-DDE to medaka d-rR strain after a one-time embryonic exposure by in ovo nanoinjection: an early through juvenile life cycle assessment.

The toxicity of o,p'-DDE (1,1-dichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl) ethylene) was evaluated in embryos of medaka (Oryzias latipes) following a one time exposure via nanoinjection. Medaka eggs (early gastrula) were injected with 0.5 nl of triolein (vehicle control) or 0.5 nl of 4 graded doses (0.0005-0.5 ng/egg) of o,p'-DDE in triolein. Embryos were allowed to develop, and fry were reared. Embryonic survival was monitored daily during the first 10 d until hatching and thereafter, on a weekly basis until day 59, at which time the fish were monitored for sexual maturity until day 107. In general, o,p'-DDE caused a dose- and time-dependent mortality. No changes in mortality were observed between the last two time points (day 38 and 59, respectively), and hence a 59 day-LD50 of 346 ng o,p'-DDE/egg was derived from the linear dose-response relationship. Prior to late stage death, only isolated cases of cardiovascular lesions and spinal deformities were observed, but were not dose-dependent. The lowest observable adverse effect level (LOAEL), based on upper 95% CI for regression line=0.0018 mg/kg, and the LOAEL based on exposure doses=0.5 mg/kg. Likewise, the no observable adverse effect level (NOAEL) based on linear extrapolation to 100% survival=0.0000388 mg/kg, while the NOAEL based on exposure doses=0.05 mg/kg. The nanoinjection medaka model has potential in the study of hormonally active compounds in the environment.

The organochlorine o,p'-DDD disrupts the adrenal steroidogenic signaling pathway in rainbow trout (Oncorhynchus mykiss).

The mechanisms of action of o,p'-DDD on adrenal steroidogenesis were investigated in vitro in rainbow trout (Oncorhynchus mykiss). Acute exposures to o,p'-DDD inhibited ACTH-stimulated cortisol secretion while cell viability decreased significantly only at the highest concentration tested (200 microM o,p'-DDD). Stimulation of cortisol secretion with a cAMP analogue (dibutyryl-cAMP) was inhibited at a higher concentration than that needed to inhibit ACTH-stimulated cortisol synthesis in cells exposed to o,p'-DDD. Forskolin-stimulated cortisol secretion and cAMP production, and NaF-stimulated cAMP production were inhibited in a concentration-dependent manner by o,p'-DDD. In contrast, basal cortisol secretion was stimulated while basal cAMP production was unaffected by o,p'-DDD. Pregnenolone-stimulated cortisol secretion was enhanced by o,p'-DDD at a physiologically relevant pregnenolone concentration, while o,p'-DDD inhibited cortisol secretion when a pharmacological concentration of pregnenolone was used. Our results suggest that the cAMP generation step is a target in o,p'-DDD-mediated disruption of ACTH-stimulated adrenal steroidogenesis in rainbow trout but that other downstream targets such as steroidogenic enzymes responsible for cortisol synthesis might also be affected.

Irreversible binding of o,p'-DDD in interrenal cells of Atlantic cod (Gadus morhua).

Precision-cut tissue slices of the anterior kidney from Atlantic cod (Gadus morhua) were prepared with a Krumdieck tissue slicer and exposed to 2-(2-chlorophenyl)-2-(4-chloro-(14C)phenyl)-1,1-dichlorethane (o,p(')-[14C]DDD) in vitro. Microautoradiography revealed irreversible o,p(')-DDD-derived binding confined to the glucocorticoid producing interrenal cells (adrenocortical analogues). This cell-selective binding was confirmed by means of autoradiography at different levels of resolution on Atlantic cod administered o,p(')-[14C]DDD intragastrically. The results provide evidence for a site-specific metabolic activation and irreversible binding of o,p(')-DDD in the interrenal cells, which, in turn, may modify glucocorticoid homeostasis.

In ovo exposure to o,p -DDE affects sexual development but not sexual differentiation in Japanese medaka (Oryzias latipes).

Despite being banned in many countries, dichlorodiphenyltrichloroethane (DDT) and its metabolites dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD) continue to be found in fish tissues at concentrations of concern. Like o,p -DDT, o,p -DDE is estrogenic and is believed to exert its effects through binding to the estrogen receptor. The limited toxicologic data for o,p -DDE suggest that it decreases fecundity and fertility of fishes. We conducted an egg injection study using the d-rR strain of medaka and environmentally relevant concentrations of o,p -DDE to examine its effects on sexual differentiation and development. The gonads of exposed fish showed no evidence of sex reversal or intersex. However, other gonad abnormalities occurred in exposed individuals. Females exhibited few vitellogenic oocytes and increased atresia. Male testes appeared morphologically normal but were very small. Gonadosomatic index values for both sexes were lower for exposed fish. Our observations of abnormal female and very small male gonads after in ovo o,p -DDE exposure may be indicative of effects on early endocrine processes important for normal ovarian and testicular development.

Impact of monitoring plasma 1,1-dichlorodiphenildichloroethane (o,p'DDD) levels on the treatment of patients with adrenocortical carcinoma.

BACKGROUND: It has been suggested recently that 1,1-dichlorodiphenildichloroethane (o,p'DDD) elicits a dose effect relation in the treatment of patients with adrenocortical carcinoma (ACC). The authors performed a single-center, prospective study with two major objectives: 1) to confirm the interest of plasma o,p'DDD level measurement as a prognostic factor of response to o,p'DDD therapy; and 2) to look for parameters associated with a therapeutic plasma o,p'DDD level, especially the daily o,p'DDD dose. METHODS: Since 1995, patients with ACC who were referred to the Gustave-Roussy Institute have been enrolled prospectively in the study. Therapy with o,p'DDD was given as first-line therapy in 13 patients with metastatic disease or as adjuvant therapy in 11 patients. Oral o,p'DDD was given in three separate doses up to at least 6-12 g per day together with substitutive adrenal therapy. Plasma o,p'DDD levels were measured using high-performance liquid chromatography every 2 months. The o,p'DDD therapy was monitored to achieve plasma o,p'DDD levels within 14-20 mg/L. World Health Organization criteria were used to evaluate tumor response and toxicity. RESULTS: Twenty-four patients with ACC were studied, and a plasma o,p'DDD level > 14 mg/L was achieved in 14 patients (58%). An objective tumor response was observed in four patients with metastatic lesions (31%): One was response was complete, and three were objective hormonal responses. These tumor responses were observed among the six patients who achieved therapeutic plasma o,p'DDD levels. In contrast, no response was observed among the seven patients with plasma o,p'DDD levels that remained consistently low. Eight of 11 patients who received o,p'DDD as adjuvant therapy had disease recurrence, although the plasma o,p'DDD level was > 14 mg/L in 6 patients. Grade 3 or 4 neurologic toxicity was observed in three patients (12%), all with an o,p'DDD level > 20 mg/L. The daily o,p'DDD dose was the only parameter associated with the highest plasma o,p'DDD trough levels: It explained 35% of the variability in the plasma o,p'DDD level. A median interval of 3.7 months was found necessary to achieve the highest o,p'DDD trough levels. CONCLUSIONS: The results confirm the prognostic impact of the plasma o,p'DDD level in patients with metastatic ACC and its interest in avoiding toxicity.

Primary culture system of adrenocortical cells from dogs to evaluate direct effects of chemicals on steroidogenesis.

The present study was conducted to confirm the usefulness of a primary culture system of adrenocortical cells from dogs for detecting the direct effects of the chemicals on adrenal cortex. Corticosteroid levels in the culture supernatant were measured using high-performance liquid chromatography (HPLC) following 24-h incubation with the chemicals. Ketoconazole, miconazole, metyrapone, aminoglutethimide, and 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloroethane (o,p-DDD), which were known to inhibit cortisol production were evaluated in this system. Both viable cells and corticosteroid levels were decreased by o,p-DDD treatment. Other chemicals showed various inhibition patterns of corticosteroid levels as follows without affecting cell viability. Ketoconazole decreased total corticosteroids level by mainly due to the decreases in cortisol and 11-deoxycortisol levels. Miconazole decreased cortisol and 11-deoxycortisol levels, however, slightly increased corticosterone level. Metyrapone decreased cortisol and corticosterone levels as 11-deoxycortisol and 11-deoxycorticosterone levels were increased. Aminoglutethimide decreased total corticosteroids level by mainly decreasing cortisol, corticosterone and 11-deoxycortisol levels. These results suggested that determination of the pattern of corticosteroid levels by HPLC in this system well reflected the mode of their action on steroidogenesis. Thus, we conclude this simple system was useful to determine the direct effects of chemicals on steroidogenesis in the adrenal cortex.

Reporter cell lines to study the estrogenic effects of xenoestrogens.

In order to characterize the estrogenic activity of chemicals, we established complementary in vitro recombinant receptor-reporter gene assays in stably transfected MCF-7 and HeLa cells. MCF-7 cells which express the endogenous estrogen receptor alpha (ER alpha) were stably transfected with only an estrogen-regulated luciferase gene. These cells enable the detection of compounds which bind to ER alpha or interfere with the induction of ER alpha mediated gene expression. Furthermore, HeLa cells, which do not express endogenous ERs, were transfected with an ER alpha or an ER beta construct together with an estrogen-regulated luciferase gene, or a chimeric GAL4-ER alpha receptor and the corresponding luciferase reporter gene. Finally, we tested these four cellular models as tools to check the estrogenic activities of several potential xenoestrogens and to detect estrogenic activity in wastewater sewage treatment effluents. In all of the models, nonylphenol mixture (NPm), 4n-nonylphenol (4nNP), 2,4'-DDE, 4,4'-DDE and wastewater sewage treatment effluent were active, while PCB mixture (Aroclor 1254), PCB 77, atrazine and lindane (gamma hexachlorocyclohexane) were inactive. Dioxin partially activates the estrogen receptor in MCF-7 cells while in HeLa-derived cell lines, it decreased the estrogenic-induced expression of luciferase.

Effects of in vitro exposures to cadmium, mercury, zinc, and 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane on steroidogenesis by dispersed interrenal cells of rainbow trout (Oncorhynchus mykiss).

Numerous environmental xenobiotics can act as endocrine disrupters in wildlife species. Fish chronically exposed to pollutants exhibit a deficiency in the synthesis of cortisol, a glucocorticosteroid hormone secreted by interrenal steroidogenic cells in response to ACTH by a mechanism mediated by cAMP. The capacity of a series of heavy metals (CdCl2, ZnCl2, HgCl2, and CH3HgCl) and 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane (o,p'-DDD) to disrupt cortisol secretion was determined in dispersed interrenal cells of rainbow trout (Oncorhynchus mykiss) exposed in vitro to the toxicant, by measuring cortisol secretion stimulated with ACTH or dibutiryl-cAMP (dbcAMP) and by assessing cell viability. The effect of cadmium in presence of zinc on the interrenal cells was also determined. The median lethal concentration (LC50, dose that kills 50% of the cells), median effective concentration (EC50, dose that inhibits cortisol secretion by 50%), and LC50/EC50 ratio were determined for each chemical to compare its endocrine toxicity and to test the specificity of the toxicants to act as endocrine disrupters. HgCl2 had the lowest EC50 and LC50; it was the most toxic of the chemicals tested. The LC50/EC50 ratio was the highest for ZnCl2 and CdCl2, indicating that these toxicants had the most specific endocrine toxicity. The mechanism of toxicity of heavy metals on cortisol-secreting cells involves a site situated downstream from the step generating cAMP, while o,p'-DDD seemed to impair a step located between adenyl cyclase activation and the ACTH binding. No evidence for a protector effect of zinc against cadmium toxicity was found.