Giovanna De Angelis

A critical review of comparative acute toxicity data on freshwater fish

Abstract An exhaustive literature survey has been carried out to collect comparative data of acute toxicity of organic chemicals to species recommended by OECD for toxicity testing. These species are: trout (Salmo gairdneri), bluegill (Lepomis macrochirus), guppy (Poecilia reticulata), carp (Cyprinus carpio), fathead min-now (Pimephales promelas), red killifish (Oryzias latipes), golden orfe (Leuciscus idus) and zebra fish (Brachydanio rerio). Comparative information was available only among the first five species. Among the 200 collected compounds, 24 (most of which were phosphoesters), showed a marked species-dependent toxicity, with 96 h LC50 ratios between two species ranging from 10 to about 300. With reference to the action of the selective toxicants, fathead minnow was the most resistant species in practically all the available comparisons. No dependence of the selective toxicity on the n-octanol/water partition coefficient of the chemicals could be observed.

Short-term effects of adolescent methylphenidate exposure on brain striatal gene expression and sexual/endocrine parameters in male rats.

Abstract:  Exposure to methylphenidate (MPH) during adolescence is the elective therapy for attention deficit/hyperactivity disorder (ADHD) children, but raises major concerns for public health, due to possibly persistent neurobehavioral changes. Rats (30‐ to 44‐days old) were administered MPH (2 mg/kg, i.p once daily) or saline (SAL). At the end of the treatment we collected plasma, testicular, liver, and brain (striatum) samples. The testes and liver were used to evaluate conventional reproductive and metabolic endpoints. Testes of MPH‐exposed rats weighed more and contained an increased quantity of sperm, whereas testicular levels of testosterone (TST) were markedly decreased. The MPH treatment exerted an inductive effect on enzymatic activity of TST hydroxylases, resulting in increased hepatic TST catabolism. These findings suggest that subchronic MPH exposure in adolescent rats could have a trophic action on testis growth and a negative impact on TST metabolism. We have analyzed striatal gene expression profiles as a consequence of MPH exposure during adolescence, using microarray technology. More than 700 genes were upregulated in the striatum of MPH‐treated rats (foldchange >1.5). A first group of genes were apparently involved in migration of immature neural/glial cells and/or growth of novel axons. These genes include matrix proteases (ADAM‐1, MMP14), their inhibitors (TIMP‐2, TIMP‐3), the hyaluronan‐mediated motility receptor (RHAMM), and growth factors (transforming growth factor‐β3 [TGF‐β3] and fibroblast growth factor 14 [FGF14]). A second group of genes were suggestive of active axonal myelination. These genes mediate survival of immature cells after contact with newly produced axonal matrix (laminin B1, collagens, integrin alpha 6) and stabilization of myelinating glia‐axon contacts (RAB13, contactins 3 and 4). A third group indicated the appearance and/or upregulation of mature processes. The latter included genes for: K+ channels (TASK‐1, TASK‐5), intercellular junctions (connexin30), neurotransmitter receptors (adrenergic alpha 1B, kainate 2, serotonin 7, GABA‐A), as well as major proteins responsible for their transport and/or anchoring (Homer 1, MAGUK MPP3, Shank2). All these genes were possibly involved in synaptic plasticity, namely the formation, maturation, and stabilization of new neural connections within the striatum. MPH treatment seems to potentiate synaptic plasticity, which is an age‐dependent developmental phenomenon that adolescent rats are very likely to show, compared to adults. Our observations suggest that adolescent MPH exposure causes only transient changes in reproductive and hormonal parameters, and a more enduring enhancement of neurobehavioral plasticity.

Early exposure to low doses of atrazine affects behavior in juvenile and adult CD1 mice

Environmental exposure to endocrine disrupting chemicals is receiving increasing attention, with particular regard to distinct periods of development where neuroendocrine circuitries are critical for shaping the mammalian brain. Atrazine (ATZ), a widely used herbicide, has been reported to affect steroid hormones and interfere with pathways critical for sex-specific physiological and behavioral development. Aim of the present study was to evaluate effects of perinatal exposure to environmentally relevant subtoxic doses of ATZ, on neurobehavioral development in mice and investigate possible alterations in steroid hormone metabolism. Neurobehavioral development of female and male mice delivered from CD1 dams, and daily exposed from Gestational Day 14 until Postnatal Day 21 (PND 21) to 1 or 100 μg/kg bw ATZ, was investigated. Specifically, locomotor and exploratory activity, social interactions and cognitive performance were evaluated at PND 16, 31 and 60, respectively. Moreover, general toxicity clinical signs, testicular parameters, rate of testosterone metabolism and aromatase activity in F1 male liver were analyzed at adulthood. Changes in exploratory profile and in affiliative/investigative behavior were observed, revealing a feminization of behavioral profile in ATZ-exposed males. Alteration in learning performance at adulthood was also evident. A limited decreased sperm count and concentration, as well as some slight impairment in hepatic testosterone metabolism and in aromatase activity (slightly but not significantly decreased) were observed in both low and high dose exposed animals. In conclusion developmental exposure to non-toxic, environmentally relevant doses of ATZ can produce subtle functional alterations, detectable in juvenile rodents by a detailed behavioral analysis. Behavioral disturbances appeared mainly related with neurodevelopmental disorder affecting the social domain and the emotional/affective repertoire, although further research is needed to elucidate the mechanism through which the effects are induced.

Xenobiotic-metabolizing enzyme systems in test fish. I. Comparative studies of liver microsomal monooxygenases.

All monooxygenase activities assayed in guppy (Poecilia reticulata) and red killifish (Oryzias latipes) were 7-10 times higher than those measured in trout (Salmo gairdneri), carp (Cyprinus carpio), and golden orfe (Leuciscus idus). Cytochrome P-450 was 3 times higher in the former fish than in all the other species. Zebra fish (Brachydanio rerio) 7-ethoxycoumarin O-deethylase and bluegill (Lepomis macrochirus) ethylmorphine N-demethylase (EMND) were not greatly different from those of the former group. alpha-Naphthoflavone (NF) and methyrapone (MET) exerted qualitatively different actions on benzo[a]pyrene hydroxylase of guppy, trout, and bluegill liver: both chemicals enhanced the bluegill activity and inhibited that of trout; the guppy liver enzyme was inhibited by MET and activated by NF. EMND activity was inhibited by either compound in the three species. The relevance of all these data to the European Economic Community ecotoxicity tests is discussed.

The food contaminant semicarbazide acts as an endocrine disrupter: Evidence from an integrated in vivo/in vitro approach

Semicarbazide (SEM) is a by-product of the blowing agent azodicarbonamide, present in glass jar-sealed foodstuffs mainly baby foods. The pleiotropic in vivo SEM toxicological effects suggested to explore its possible role as endocrine modulator. Endocrine effects of SEM were assessed in vivo in male and female rats after oral administration for 28 days at 0, 40, 75, 140mg/kg bw pro die during the juvenile period. Vaginal opening and preputial separation were recorded. Concentration of sex steroid in blood, the ex vivo hepatic aromatase activity and testosterone catabolism were detected. The in vitro approach to test SEM role as (anti)estrogen or N-methyl-d-aspartate receptors (NMDARs)-(anti)agonist included different assays: yeast estrogenicity, MCF-7 proliferation, stimulation of the alkaline phosphatase activity in Ishikawa cells and LNCaP-based NMDAR interference assay. In vivo SEM-treated female rats showed delayed vaginal opening at all tested doses, whereas in males preputial separation was anticipated at SEM 40 and 75mg/kg and delayed at 140mg/kg, the latter effect probably due to the significantly decreased body weight gain seen at the higher dose in both sexes. Serum estrogen levels were dose-dependently reduced in treated females, whereas dehydrotestosterone serum levels were also decreased but a clear dose-response was not evidenced. Testosterone catabolism was altered in a gender-related way, aromatase activity was increased in treated males at 75 and 140mg/kg and in females in all dose groups. In the three estradiol-competitive assays, SEM showed a weak anti-estrogenic activity, whereas in the LNCaP-based NMDAR interference assay SEM activity resembled MK-801 antagonist effect. SEM appeared to act as an endocrine disrupter showing multiple and gender specific mechanisms of action(s). A possible cascade-mechanism of SEM on reproductive signalling pathways may be hypothesized. Such in vivo-in vitro approach appeared to be an useful tool to highlight SEM activity on endocrine homeostasis.

Xenobiotic metabolizing enzyme systems in test fish: III. Comparative studies of liver cytosolic glutathione S-transferases

The reduced glutathione (GSH)-conjugating capacities of the livers of trout, carp, zebra fish, guppy, and bluegill have been evaluated with several substrates preferentially metabolized by the major GSH-transferase isozymes. Carp and zebra fish lack GSH-transferase activity with 1,2-epoxy-3-(p-nitrophenoxy)propane. Guppy is characterized by the highest activities with 1-chloro-2,4-dinitrobenzene and the absence of activity toward trans-4-phenyl-3-buten-2-one. Enzyme activities with the other substrates demonstrate quantitative interspecific differences which presumably are of minor relevance to the metabolism and toxicity of chemicals.

Foetal and neonatal exposure to chlorpyrifos: biochemical and metabolic alterations in the mouse liver at different developmental stages.

The mechanisms implicated in the age-related toxicity, including its neurobehavioral effects after subtoxic developmental exposure to chlorpyrifos (CPF), a widely used insecticide, have not been fully elucidated yet. With the aim of investigating whether metabolic differences during ontogeny could account for the age-related susceptibility to CPF, we examined the developmental time-course of hepatic metabolizing enzymes and CPF metabolism in a cohort of mice exposed either prenatally (gestational day 15-18) and/or postnatally (postnatal day (PND) 11-14) to CPF at doses which were previously reported to induce neurobehavioural alterations, in the absence of brain acetyl-cholinesterase inhibition. Testosterone hydroxylase activity, CPF ex vivo biotransformation, glutathione content, as well as aromatase activity were determined in the liver of control and treated male and female mice at PND0, 9, 15 and 150. In control mice most Cyp activities were detectable and progressively increased up to PND15. In newborn control mice CPF bioactivation was much higher than the Cyp-catalysed detoxication, negligible at birth, indicating a possible increased susceptibility to CPF-induced effects in newborn mice. Detoxication rapidly increased with age, so that Cyp-related metabolic features cannot explain the higher susceptibility of juvenile mice. The observed age-dependent metabolic picture was partially altered by CPF prenatal treatment. Following in utero exposure CPF detoxifying capability was enhanced at birth and reduced at PND15, when CPF-oxon formation was slightly increased. No effects were evident at adulthood. Prenatal dosing was more effective in causing metabolic alterations than CPF postnatal treatment; no potentiation was observed in mice experiencing pre- plus post-natal CPF administration. Both in utero and postnatal CPF exposure decreased aromatase activity by 50% at PND9 and 15; this effect together with the presence of higher levels of the sex-specific Cyp2c activity at adulthood in male mice may suggest the occurrence of long-lasting impairment in the expression of hepatic Cyps under hormonal regulation. Altogether, the alterations in CPF Cyp-mediated biotransformation caused by perinatal CPF exposure seem not sufficient per se to explain the reported vulnerability of developing central nervous system to this insecticide, which can be due also to the parent compound itself or to the activation of different toxicological pathways. The hypothesis that observed effects on aromatase and sex-specific Cyp activity may be associated with a possible interference with the long-term alterations in sex-specific behavioural pattern deserves further investigation.

Correlation of a specific mitochondrial phospholipid-phosgene adduct with chloroform acute toxicity.

The dose and time dependence of formation of a specific adduct between mitochondrial phospholipid and phosgene have been determined in the liver of Sprague-Dawley (SD) rats as well as in the liver and kidney of B6C3F1 mice after dosing with chloroform. Rats were induced with phenobarbital or non-induced. Determination of tissue glutathione (GSH) and of serum markers of hepatotoxicity and nephrotoxicity was also carried out. With dose-dependence experiments, a strong correlation between the formation of the specific phospholipid adduct, GSH depletion and organ toxicity could be evidenced in all the organs studied. With non-induced SD rats, no such effects could be induced up to a dose of 740 mg/kg. Time-course studies with B6C3F1 mice indicated that the specific adduct formation took place at very early times after chloroform dosing and was concurrent with GSH depletion. The adduct formed during even transient GSH depletion (residual level: 30% of control) and persisted after restoration of GSH levels. Following a chloroform dose at the hepatotoxicity threshold (150 mg/kg), the elimination of the adduct in the liver occurred within 24 h and correlated with the recovery of ALT, which was slightly increased (12 times) after treatment. Following a moderately nephrotoxic dose (60 mg/kg), the renal adduct persisted longer than 48 h, when a 100% increase in blood urea nitrogen and a 40% increase in serum creatinine indicated the onset of organ damage. The formation of the adduct in the liver mitochondria of B6C3F1 mice was associated with the decrease of phosphatidyl-ethanolamine (PE), in line with previous results in rat liver indicating that the adduct results from the reaction of phosgene with PE. The adduct levels implicated the reaction of phosgene with about 50% PE molecules in the liver mitochondrial membrane of phenobarbital-induced SD rats and of about 10% PE molecules of the inner mitochondrial membrane of the liver of B6C3F1 mice. The association of this adduct with the toxic effects of chloroform makes it a very good candidate as the primary critical alteration in the sequence of events leading to cell death caused by chloroform.

Effect of lindane on CYP-mediated steroid hormone metabolism in male mice following in utero exposure.

A wide number of pesticides, including highly persistent organochlorinated compounds, such as lindane (LIN), may induce reproductive and developmental alterations by directly binding to the estrogen/androgen receptors or altering steroid hormone metabolism. In the present work, we have investigated whether LIN in utero exposure of CD1 mice affects the reproductive system in male offspring by causing an impairment of the CYP‐dependent steroid hormone metabolism. Dam exposure to 25 mg kg−1 b.w. LIN occurred during critical developmental periods, from gestational days 9 to 16. Effects on hepatic CYP‐mediated testosterone (TST) hydroxylase, aromatase activities and testicular parameters were tested at postnatal days (PND 50, 65–69, 100) that are critical for sexual maturation in CD1 mice. In the adult F1 mice significant changes of male reproductive endpoints (testis weight, spermatid number) as well as dramatic effects on CYP‐mediated TST metabolism were observed on PND 65–69, in the absence of any of systemic toxicity. The levels of TST 6β‐ and 2α‐hydroxylation and dehydrogenation showed the highest level of reduction, suggesting CYP 3A and 2C families as the major target of LIN induced effects. All changes were almost recovered on PND 100. No effects on aromatase activity were evidenced. Overall, these findings provide useful information for a better characterization of the LIN mode of action. They suggest that LIN‐induced toxicity in males is linked to an impairment of steroid hormone homeostasis, due to CYP‐mediated TST catabolism modulation and differs from LIN receptor‐mediated mechanism previously reported in females. Copyright

Xenobiotic-metabolizing enzyme systems in test fish. II: The ethylmorphine N-demethylase activity of guppy (Poecilia reticulata) liver

The oxidative demethylation of the model substrate ethylmorphine has been characterized for the first time in the liver of a fish (Poecilia reticulata). The enzyme showed maximal activity at 35 degrees C and pH values higher than 8. The values of Km and Vmax for the reaction were 0.83 +/- 0.11 mM and 4.64 +/- 0.81 nmol HCHO/(mg microsomal protein) per min. The activity is attributed to the cytochrome P-450-dependent monoxygenase system, since it is inhibited by CO and requires NADPH; moreover it is inhibited competitively by alpha-naphthoflavone and non-competitively by metyrapone. The enzyme activity is induced by a two-week treatment of fish with phenobarbital and may be associated with a protein band of Mr 54,000.