Evaluating the evidence for non-monotonic dose-response relationships: A systematic literature review and (re-)analysis of in vivo toxicity data in the area of food safety.

This study aims to evaluate the evidence for the existence of non-monotonic dose-responses (NMDRs) of substances in the area of food safety. This review was performed following the systematic review methodology with the aim to identify in vivo studies published between January 2002 and February 2015 containing evidence for potential NMDRs. Inclusion and reliability criteria were defined and used to select relevant and reliable studies. A set of six checkpoints was developed to establish the likelihood that the data retrieved contained evidence for NMDR. In this review, 49 in vivo studies were identified as relevant and reliable, of which 42 were used for dose-response analysis. These studies contained 179 in vivo dose-response datasets with at least five dose groups (and a control group) as fewer doses cannot provide evidence for NMDR. These datasets were extracted and analyzed using the PROAST software package. The resulting dose-response relationships were evaluated for possible evidence of NMDRs by applying the six checkpoints. In total, 10 out of the 179 in vivo datasets fulfilled all six checkpoints. While these datasets could be considered as providing evidence for NMDR, replicated studies would still be needed to check if the results can be reproduced to rule out that the non-monotonicity was caused by incidental anomalies in that specific study. This approach, combining a systematic review with a set of checkpoints, is new and appears useful for future evaluations of the dose response datasets regarding evidence of non-monotonicity.

Does EU legislation allow the use of the Benchmark Dose (BMD) approach for risk assessment?

Hazard characterisation is largely based on an approach of (statistically) comparing dose groups with the controls in order to derive points of departure such as no-observed-adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs). This approach suggests the absence of any relevant effect at the NOAEL. The NOAEL approach has been debated for decades. A recent Scientific Opinion by the European Food Safety Authority (EFSA) concluded that the Benchmark Dose (BMD) approach should be preferred over the NOAEL approach for deriving human (health-based) limit or guidance values. Nonetheless, the BMD approach is used infrequently within European regulatory frameworks. The reason for this may lie in legislation or guidelines requiring the use of the NOAEL approach. In this context, various EU regulatory frameworks were examined on such demands. Interestingly, no single legislation was identified containing statutory requirements in conflict with the use of the BMD approach.

Role of biokinetics in risk assessment of drugs and chemicals in children.

Whether children incur different risks from xenobiotics than adults will depend on the exposure, biokinetics, and dynamics of compound. In this paper, current knowledge on developmental physiology and possible effects on biokinetics are evaluated and the role of biokinetics in risk assessment both for drugs and chemicals is discussed. It is concluded that most dramatic age-related physiological changes that may affect biokinetics occur in the first 6-12 months of age. The difference in internal exposure between children and adults can generally be predicted from already known developmental physiological differences. However, for risk assessment it will also be necessary to determine whether internal exposure is within the drug's therapeutic window or if it will exceed the NOAEL of a chemical. Furthermore, the effects of internal exposure of potentially harmful compounds on developing organ systems is of utmost importance. However, knowledge on this aspect is very limited. Risk assessment in children could be improved by: (1) application of pediatric PBPK-models in order to gain insight into internal exposure in children, (2) studies in juvenile animals for studying effects on developing systems, and (3) extrapolation of knowledge on the relationship between internal exposure and dynamics for drugs to other chemicals.

Hyperresponsiveness to phencyclidine in animals lesioned in the amygdala on day 7 of life. Implications for an animal model of schizophrenia.

Phencyclidine (PCP) has been described to exacerbate psychotic symptoms in patients suffering from schizophrenia. In rats, PCP, dose-dependently, induces hyperactivity, stereotyped behaviour and social isolation, postulated to represent the positive (hyperactivity, stereotypy) and negative (social isolation) symptoms of schizophrenia. Based on previous studies, ibotenic acid lesions in the amygdala on day 7 of life have been proposed as an animal model of psychiatric neurodevelopmental disorders like schizophrenia. The purpose of the present study was to determine whether the responsiveness to PCP on locomotor activity in animals lesioned in the amygdala on day 7 of life is different from the response to this drug in sham-operated animals. The effect of graded doses of PCP on behaviour was assessed in a small open field. Animals lesioned in the amygdala on day 7 of life appeared to be hyperresponsive to PCP compared to sham-operated animals. The hyperresponsiveness to PCP in rats lesioned in the amygdala on day 7 of life further contributes to the validation of this putative animal model of schizophrenia.

Adaptation and habituation to an open field and responses to various stressful events in animals with neonatal lesions in the amygdala or ventral hippocampus.

A rat model of neurodevelopmental psychopathological disorders, designed to determine neurodevelopmental deficits following damage to the brain early in life, was used to investigate behavioural changes in adaptation and habituation to an open field and responses to different kinds of stressful events. Animals with bilateral ibotenic acid lesions in the amygdala or ventral hippocampus on day 7 or 21 of life were compared to sham-operated animals. According to the model it was assumed that behavioural changes in animals lesioned on day 7, but not in animals lesioned on day 21 of life, were caused by maldevelopment of one or more structures connected to the damaged area. Animals lesioned in the amygdala or ventral hippocampus on day 7, but not animals lesioned in these structures on day 21 of life, displayed decreased (within-session) adaptation and (between-session) habituation to the open field and a decrease in immobility in the forced swim test, whereas only animals lesioned in the amygdala displayed enhanced general activity. These results were indicative of neurodevelopmental deficits. No changes in stress-induced hyperthermia were found, while animals lesioned in the amygdala both on day 7 or 21 of life exhibited decreased conditioned ultrasonic vocalizations. These latter results suggest that the amygdala is implicated in the conditioned stress-induced response. The contribution of the present findings to the animal model of neurodevelopmental disorders like schizophrenia and possible brain structures and neurotransmitter systems involved in the neurodevelopmental deficits are discussed.

Early amygdala damage in the rat as a model for neurodevelopmental psychopathological disorders.

Neurodevelopmental disorders in medial temporal lobe structures may underlie psychopathological diseases such as schizophrenia and autism. To construct an animal model for these developmental disorders, social and non-social behavioural responses were assessed in rats with ibotenic acid lesions of the (baso-)lateral and central amygdala or ventral hippocampus, induced early in life. Lesioning the amygdala on day 7 after birth resulted in a variety of behavioural disturbances later in life, whereas after similar lesions on day 21 after birth no disturbances developed, except for deficits in social behaviours. Lesioning the hippocampus led to much less disturbances. The results show that amygdala and hippocampus damage at a specific point early in life results in enduring behavioural disturbances that become more manifest after puberty. In particular, lesions of the amygdala on day 7 of life may serve as a rat model with face and construct validity for neurodevelopmental disorders in studying psychopathology.

Neutrophil intracellular pH and phagocytosis after thermal trauma.

Severe burn trauma induces an acquired dysfunction of neutrophil granulocytes. As neutrophil function is considerably influenced by intracellular pH (pH(i)), the pH(i) of blood neutrophils was longitudinally determined in 19 patients with major burns. pH(i) was measured by a flow cytometric method using the pH-sensitive fluoroprobe carboxy-semi-naphthorhodafluor-1; mechanisms influencing the pH(i) were examined by addition of amiloride (inhibition of Na(+)/H(+) countertransport), diphenylene iodonium (inhibition of NADPH oxidase) and N-formyl-methionyl-leucyl-phenylalanine (activation of H(+) extrusion). The neutrophil phagocytic activity was measured in parallel. Patients showed distinct alterations of neutrophil pH(i), depending on whether they developed sepsis in the postburn period or not. In the sepsis patients pH(i) did not deviate from the values found in healthy volunteers in the first days after injury, but rose afterwards, with significant intracellular alkalinization in the second postburn week (P<0.05). In contrast, patients without sepsis had increased pH(i) in the first (P<0.01 at days 1-2), but not in the second week after burn trauma. Inhibition studies showed that postburn intracellular alkalinization is not solely caused by activation of Na(+)/H(+) countertransport. A clear relation between pH(i) changes and phagocytosis could not be established.

Emotional and footshock stimuli induce differential long-lasting behavioural effects in rats; involvement of opioids.

Rats were exposed to either a footshock stimulus (FS) or emotional stimulus (ES, forced perception of another rat receiving footshocks) during a daily 10-min session for 5 consecutive days. The consequences of FS and ES on their behavioural responsiveness were assessed at different post-stress intervals using a small open-field. FS induced a decrease in ambulation, rearing and sniffing and an increased immobility in the small open field. These effects were present in rats tested immediately after the last session and remained present for at least 15 days. In contrast, ES induced a transient decrease in ambulation and rearing immediately after the last session, but in the period from half an hour until at least 15 days after the stimulus experience, an increase in ambulation, rearing and sniffing was observed. Exposure to one footshock per session for 5 consecutive days or to 10 footshocks in a single session also resulted in a long-lasting reduction in ambulation and sniffing and an increase in immobility. The former regime did not influence the behavioural response of ES rats, but the latter resulted in an increase in ambulation, rearing and sniffing in ES rats. Naloxone (1 mg/kg s.c.) pretreatment antagonized the increased behavioural activity of the ES rats whereas the activity of control and FS animals was not affected, suggesting an involvement of endogenous opioid systems in the behavioural responses observed in ES rats. It is suggested that the behavioural responses of the ES and FS animals are regulated by different mechanisms.

A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons.

The mesencephalic dopaminergic (mesDA) system regulates behavior and movement control and has been implicated in psychiatric and affective disorders. We have identified a bicoid-related homeobox gene, Ptx3, a member of the Ptx-subfamily, that is uniquely expressed in these neurons. Its expression starting at E11.5 in the developing mouse midbrain correlates with the appearance of mesDA neurons. The number of Ptx3-expressing neurons is reduced in Parkinson patients, and these neurons are absent from 6-hydroxydopamine-lesioned rats, an animal model for this disease. Thus, Ptx3 is a unique transcription factor marking the mesDA neurons at the exclusion of other dopaminergic neurons, and it may be involved in developmental determination of this neuronal lineage.

Modifications in glutamatergic transmission after dopamine depletion of the nucleus accumbens. A combined in vivo/in vitro electrophysiological study in the rat.

The interaction between the glutamatergic and dopaminergic input in the nucleus accumbens was examined by studying the effects of dopamine depletion of the nucleus accumbens on the local field potentials, and the L-glutamate elicited responses of the nucleus accumbens in anaesthetized rats in vivo. A characteristic field potential in the nucleus accumbens is evoked by electrical stimulation of the fornix/fimbria fibres, with a monosynaptic positive peak at 10 ms (P10). Rats were unilaterally injected with 6-hydroxydopamine in the nucleus accumbens. The contralateral accumbens was sham lesioned. The rats were divided into short-term and long-term survival groups of one to two weeks and 24 weeks, respectively. In the short-term group, a striking increase (up to three times) of the amplitude of the P10 components, at the site of the lesion, compared with the sham lesioned contralateral accumbens and untreated rats, was found. The long-term group could still display a slight increase although on average this was not significantly different from controls. In the short-term group, at the centre of the lesion, the paired-pulse facilitation ratio was significantly smaller than at the more ventral, less denervated, border of the accumbens. These differences were no longer visible in the long-term group. Single-unit activity of the accumbens, elicited by the iontophoretical application of L-glutamate showed, in controls, a maximal firing frequency ranging from 5 to 40 Hz (mean 25 Hz), whereas in the short-term group more than 50% of the accumbens neurons fired with higher frequencies, reaching up to 90 Hz (mean 55 Hz). In the long-term group the firing frequency varied from 5 to 60 Hz (mean 41 Hz). No changes in threshold ejection glutamate current were found for both lesioned groups. In control rats the L-glutamate elicited responses of six cells tested could be suppressed by dopamine whereas in lesioned rats three of the six cells tested were unresponsive to dopamine. Intracellular recordings of accumbens cells in slices in 6-hydroxydopamine and sham lesioned rats, showed no significant changes in the intrinsic membrane properties, e.g. resting membrane potential, input resistance, spike threshold, action potential amplitude or duration. We conclude that dopamine denervation leads to an increase of excitability of the principal accumbens neurons. This is reflected by the increase of the firing frequency of these cells and of the amplitude of the evoked field potentials. The former is more likely of postsynaptic origin whereas the latter may also have a presynaptic contribution. These effects cannot be attributed to changes in intrinsic membrane properties of the cells.

The behavioral effects of habituation and challenge with apomorphine after 6-OHDA lesioning of the nucleus accumbens in rats.

In rats the function of the dopamine system in the nucleus accumbens was tested after 6-OHDA lesioning of this brain area and after ORG 2766 induced facilitation of recovery in 6-OHDA lesioned animals. A low dose of systemically administered apomorphine (50 micrograms/kg) decreased motility when sham operated rats were placed in a novel environment. A similar decrease was found in saline treated rats tested for the second time 1 day later. In thus habituated animals, the low dose of apomorphine did not induce hypomotility. Thus habituation and hypomotility after a low dose of apomorphine may be due to a similar mechanism, viz. diminished dopamine release. A higher dose of apomorphine (125 micrograms/kg) increased motility, but only when the rats were habituated to the test environment. Animals with a bilateral 6-OHDA lesion of the nucleus accumbens showed hypomotility when tested for the first time 1 week after the lesion. The low and the higher dose of apomorphine elicited hypermotility in both nonhabituated and habituated lesioned rats. Their activity was higher than in sham operated animals, suggesting supersensitivity of postsynaptically located dopamine receptor systems in lesioned rats. Treatment with the ACTH(4-9) analog ORG 2766 during the first week after induction of the lesion counteracted the hypomotility of the lesioned rats. Furthermore ORG 2766 enhanced the supersensitivity as revealed by challenge with the low dose of apomorphine.

Prolyl-leucyl-glycinamide, thyrotropin-releasing hormone and beta-endorphin-(10-16), like antidepressants, antagonize melatonin-induced behavioural changes in rats.

beta-Endorphin-(10-16), as well as a variety of antidepressants, has been reported to block the behavioural changes induced by injecting melatonin into the nucleus accumbens. In the present study the influence of subcutaneously administered prolyl-leucyl-glycinamide (PLG) and thyrotropin-releasing hormone (TRH) on the behavioural changes induced by melatonin administration in the nucleus accumbens were investigated and compared with that of beta-endorphin-(10-16). PLG and TRH were found to be as effective as beta-endorphin-(10-16) in counteracting the melatonin-induced behavioural changes. The data suggest that these peptides may serve as a starting point for the development of a new class of antidepressants.

Regulation of masculine sexual behavior: involvement of brain opioids and dopamine.

In recent years much has become known about the substrates in the brain involved in the regulation of masculine sexual behavior and the involvement of specific neurochemicals in these brain areas. In the present paper the experimental data concerning the involvement of a number of brain areas in sexual behavior are reviewed, in relation to an incentive motivational theory of sexual behavior. The review is restricted to the involvement of opioids and dopamine, of which the role in sexual motivation and behavior is best documented. Opioids in the medial preoptic area (mPOA) impair sexual performance, although the endogenous opioids systems may be quiescent in normal, sexually active rats. Dopamine in the mPOA has a facilitative role in the masculine sexual performance. The corticomedial amygdala is involved in processing of sensory information, especially olfactory stimuli, which are subsequently directed towards the mPOA. Local beta-endorphin infusion interferes with this processing. Endogenous opioids in the ventral tegmental area activate the mesoaccumbens dopamine system and stimulate the sexual motivation. Increased dopamine transmission in the nucleus accumbens correlates with increased sexual motivation and vice versa. The basolateral amygdala plays an essential role in the association of environmental stimuli with reward and therefore in the expression of conditioned sexual motivation. Finally, the reviewed data are integrated and a comprehensive view on the relations between various neural substrates is composed.

Behavioral studies on the putative gamma-type endorphin receptor using different antibodies.

To investigate the significance of endogenous, neuroleptic-like gamma-type endorphins and their putative receptors, polyclonal and monoclonal antibodies against gamma-type endorphins, which may bio-inactivate the ligands for the receptors, and monoclonal anti-idiotype antibodies, which presumably bind to the receptors, were injected into the nucleus accumbens of the rat brain. The desenkephalin-gamma-endorphin-induced antagonism of the hypomotility response elicited by challenge with apomorphine injected into the nucleus accumbens was used as test system. Both the anti-desenkephalin-gamma-endorphin antibodies and anti-idiotype antibodies blocked the action of exogenous desenkephalin-gamma-endorphin. Thus, the anti-idiotype antibodies may serve as receptor antagonists. Chronic treatment (injection into the nucleus accumbens) with the anti-idiotype antibodies induced sustained hypermotility, decreased habituation and impaired passive avoidance behavior. In such treated animals local treatment with apomorphine did not elicit hypomotility. It is suggested that gamma-type endorphins influence the setpoint for feedback regulation in dopaminergic neurons equipped with gamma-type endorphin receptor systems.

Subchronic treatment with the neuroleptic-like peptide desenkephalin-gamma-endorphin may decrease dopaminergic neurotransmission in the nucleus accumbens of rats.

In rats, subchronic administration of desenkephalin-gamma-endorphin (DE gamma E) into the nucleus accumbens or subcutaneously for 10 days resulted in hypoactivity. Intra-accumbens administration caused a significant reduction in the nucleus accumbens tissue levels of the dopamine (DA) metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Systemic administration of DE gamma E decreased DOPAC and 5-hydroxyindoleacetic acid (5-HIAA) levels in nucleus accumbens tissue. Subchronic subcutaneous DE gamma E treatment reduced the basal release of [3H]DA from rat nucleus accumbens slices in vitro and the basal release of endogenous DA and DOPAC in vivo as assessed with on-line dialysis in the nucleus accumbens of freely moving rats. The DA agonist N,N-dipropyl-7-hydroxy-2-aminotetralin (DP-7-ATN) was equally effective in inhibiting [3H]DA release elicited by electrical stimulation from slices of subchronically DE gamma E and placebo treated rats. Administration of a small dose of apomorphine caused similar reductions of the in vivo release of DA and DOPAC in both placebo and DE gamma E treated rats. These results indicate that subchronic DE gamma E treatment may decrease dopaminergic neurotransmission in the nucleus accumbens. This effect is probably not due to alterations in the sensitivity of presynaptically located DA autoreceptors mediating DA release in vitro and in vivo.

Lack of evidence for an involvement of nucleus accumbens dopamine D1 receptors in the initiation of heroin self-administration in the rat.

The involvement of dopamine D1 receptor systems in the reinforcing properties of opiate reward was studied by examining the effect of the dopamine D1 antagonist SCH23390 on the initiation of heroin self-administration in rats. The D1 antagonist was administered daily systemically or locally in the nucleus accumbens (NAC), after which the animals were allowed to self-administer heroin (IV) in a 3-h session for 5 consecutive days. Systemic treatment with SCH23390 (0.17 and 0.5 mg.kg-1) significantly decreased heroin intake during initiation of heroin self-administration, while a dose of 0.06 mg.kg-1 was not effective. Local administration of SCH23390 (0.5 and 2.5 micrograms/site) in the NAC did not affect heroin intake. Both systemic and intra-accumbal administration of SCH23390 dose dependently decreased motor behavior measured in a small open field. The attenuation of heroin intake during initiation of heroin self-administration by blockade of dopamine D1 receptor systems may be due to a decrease in the reinforcing effects of heroin or more likely to a reduction in non-reinforcement-related behavior. The dopamine D1 receptors present in the NAC are probably not involved in opiate reward.

Cholecystokinin-dopamine interactions within the nucleus accumbens in the control over behaviour by conditioned reinforcement.

Cholecystokinin (CCK) is colocalised with dopamine in the postero-medial nucleus accumbens (NAS). We have utilised an acquisition of a new response procedure to investigate the interaction between CCK and dopamine in the control over behaviour by conditioned reinforcers. A conditioned reinforcer (CR) may be defined as an initially neutral stimulus which gains control over behaviour through selective association with a primary reinforcer. Here, rats learned to associate a light/noise compound stimulus with the imminent availability of 10% sucrose reinforcement. Later, in the absence of sucrose, responding on one of two novel levers (the CR lever) was acquired and maintained by contingent presentation of the CR alone, while responding on the second lever had no programmed consequences. In Expt. 1, infusion of 10 micrograms D-amphetamine within the postero-medial NAS enhanced responding selectively on the CR lever. Infusion of sulphated CCK octapeptide (CCK: 1 or 10 ng) alone within the same area had no effect on response rate. However, infusion of CCK immediately prior to D-amphetamine caused a dose-dependent potentiation of the impact of D-amphetamine upon rates of response on the CR lever. In Expt. 2, infusion of D-amphetamine (10 micrograms) within the postero-medial NAS again enhanced responding selectively upon the CR lever. Intra-accumbens infusion of CCK (10 ng), or s.c. administration of the CCKA receptor antagonist devazepide had no effect upon response rates. However, CCK again potentiated the D-amphetamine-induced increase in rates of response, and this potentiation was blocked by pretreatment with devazepide. These results are discussed in terms of the co-modulation by CCK and dopamine of the processing of reward-related stimuli within the NAS.

Relative roles of ventral striatal D1 and D2 dopamine receptors in responding with conditioned reinforcement.

Several experiments investigated the involvement of D1 and D2 dopamine receptors in the ventral striatum in the control over behaviour by a conditioned reinforcer using an acquisition of new response procedure. Intra-accumbens infusion of either the D1 receptor antagonist, SCH 23390, or the D2 receptor antagonist, raclopride, completely blocked the potentiative effects of intra-accumbens d-amphetamine on responding with conditioned reinforcement and reduced responding to control levels. SCH 23390 was more potent than raclopride. At higher doses in the absence of d-amphetamine, both antagonists also blocked the preference for responding on the lever producing the conditioned reinforcer. Intra-accumbens infusions of either the D1 receptor agonist, SKF 38393, or the D2/3 receptor agonist, LY 171555 (quinpirole), selectively potentiated responding on the lever producing the conditioned reinforcer. Various combined infusions of the D1 and D2 agonists in specific low doses had additive, but not synergistic, effects on responding with conditioned reinforcement. None of the drugs affected the drinking of water in deprived subjects when infused intra-accumbens. These results suggest that both D1 and D2 receptors in the nucleus accumbens are involved in mediating the effects of dopamine in potentiating the control over behaviour by conditioned reinforcers.

Short duration of retroactive facilitation of social recognition in rats.

Adult rats spent less time investigating the same juvenile during a second dyadic encounter session. This decrease served as an index for social recognition. Social recognition was not influenced by isolating the juveniles for 7 days prior to experimentation. Retroactive facilitation of social recognition was observed when the two rats were confronted for a longer period of time on a given day by multiple testing. However, this facilitation was not observed after a 24-h interexposure interval between encounter sessions, even when different housing conditions during that time were taken into account, and animals were tested during 5 consecutive days. It is suggested that social recognition may be a form of short-term memory.

The ACTH-(4-9) analogue ORG 2766 facilitates denervation supersensitivity after a unilateral 6-OHDA lesion of the corpus striatum in rats.

UNLABELLED: Direct bilateral 6-OHDA lesioning of the nucleus accumbens causes a temporary reduction in motility, followed by a spontaneous recovery in 3-4 weeks. The ACTH-(4-9) analogue ORG 2766 shortens this period to 1 week. The functional and the peptide-induced facilitation of recovery are accompanied by enhanced motility upon administration of the dopamine agonist apomorphine which may be related to denervation supersensitivity. The present experiments were performed to investigate the interaction between ORG 2766 and denervation supersensitivity in another dopaminergic terminal area i.e. the corpus striatum. After a unilateral 6-OHDA lesion of the right corpus striatum, contralateral rotation was observed upon administration of a high dose of apomorphine 2, 3 and 4 weeks after the lesion, indicating supersensitivity of postsynaptic dopaminergic receptor systems. Contralateral rotation upon administration of this dose of apomorphine was observed in ORG 2766 treated animals, already at 1 week after the lesion. Peptide treatment resulted in an enhanced sensitivity for apomorphine, since contralateral rotation was observed in peptide but not in placebo treated, 6-OHDA lesioned animals after a low dose of apomorphine. IN CONCLUSION: treatment with ORG 2766 facilitates the development of denervation supersensitivity and enhances sensitivity for apomorphine probably through an increased affinity of dopaminergic receptors for dopamine agonists.