Jump to the "Prevention of cancer chemotherapy-induced toxicity" section.

Research in my laboratory is mainly focused on a group of naturally occurring chemicals called trace amines, the proteins (receptors) that detect their presence, and how these are involved in the control of cellular activity in human health and disease. In particular trace amines are thought to play a role in controlling nerve cell activity in the brain, but may also have effects in other tissues. A separate project is examining ways in which the toxicity, in particular hair loss, associated with cancer chemotherapy can be prevented. We have also previously been involved with a project to analyze the pigment and residue composition from Mesoamerican ceramic artefacts, although this project is currently on hold.

Trace Amines and Their Receptors

Trace amines are naturally occurring chemicals that are synthesized in nerve cells. The major trace amines are 2-phenylethylamine, p-tyramine and tryptamine. The thyroid hormone metabolite 3-iodothyronamine may also act as a trace amine. The synthetic route of trace amines is very similar to that of the classical monoamine neurotransmitters dopamine, noradrenaline and 5-HT. Unlike the neurotransmitters, however, trace amines are only present in very small quantities. A family of proteins (receptors) that are selectively activated by trace amines has been discovered and named Trace Amine-Associated Receptors (TAAR).

Drug Abuse and Psychiatric Disorders

TAAR are found throughout the brain, and one type, TAAR1, appears to
regulate the activity of dopamine neurons and receptors. TAAR1 is a target for a number of drugs of abuse such as methamphetamine, LSD and “ecstasy” (3,4-methylenedioxy-methamphetamine or MDMA), and through their interaction with the dopamine system TAAR1 appear to regulate the susceptibility to self administration of these psychotropic agents as well as other addictive compounds such as cocaine and alcohol. TAAR1 are being actively investigated as a novel target for the development of improved therapies for drug abuse/addiction, schizophrenia and other psychiatric disorders.

Olfaction and Species-Specific Social Cues

In addition to being present in the brain, TAAR have been validated as a new class of olfactory receptor. The one exception to this is TAAR1 which is not found in olfactory tissue. There is a large variation between species in the sub-types of TAAR that are present, with some TAAR thought to only occur in an individual species. Recent studies have identified 2-phenylethylamine as one of the chemical cues in carnivore urine that is detected by prey species. This detection is due to the presence TAAR4 in the olfactory epithelium and induces innate avoidance behaviour in prey species. A number of other naturally occurring compounds have been shown to activate other olfactory TAAR, with some of these also inducing innate behavioural responses. The role of TAAR in species-specific olfactory-mediated social cues is a growing area of research interest.

Trace Amines and TAAR Outside the Brain

The enzymes for the synthesis and degradation of trace amines are also found in a number of tissues outside of the brain. These peripheral tissues often contain one, or more, types of TAAR. In particular TAAR1 (and the trace amine synthetic machinery) is expressed in tissues central to the control of energy metabolism, including pancreatic β-cells, liver and fat cells. The function of trace amines and TAAR in these cell types, however, remains largely unknown.

Trace Amine Work in the Berry Lab

Previous work had suggested that trace amines are not stored in nerve endings, have a very short half-life (i.e. are constantly being synthesized and degraded) with ‘release’ occurring by simple diffusion. Using a synthetic system called Fluorosomes® we have directly measured the ability of trace amines and their related monoamine neurotransmitters to diffuse across lipid bilayers.

	Permeability Coefficient (Å/sec)	Diffusion half-life (sec)
p-Tyramine	22.6 ± 4.3	13.5 ± 4.1
Dopamine	7.7 ± 1.2	35.9 ± 7.0
Tryptamine	33.2 ± 3.3	6.8 ± 0.7
5-HT	4.8 ± 0.6	48.2 ± 5.9

These studies confirmed that in the absence of membrane proteins, trace amines cross lipid bilayers significantly quicker than do neurotransmitters. As part of these studies, in collaboration with Dr. Bruno Tomberli ,we developed Molecular Dynamics based computer simulation techniques to predict the lipid bilayer permeability of trace amines (or indeed any small molecule).

Using fully functional brain neuron membrane preparations (synaptosomes) we confirmed that trace amines also cross these native membranes more readily than do neurotransmitters. Surprisingly, however, when neuronal activity in the synaptosomes was simulated by depolarizing the membrane, trace amine release appeared to decrease. This is the opposite of what is seen with neurotransmitters where the vesicle stores of neurotransmitter are released in response to depolarization.

We interpret these results as indicating that trace amine release does not occur by exocytosis, and that one or more membrane transporters are activated by membrane depolarization and actively pump released trace amines back into the nerve terminal. Neuronal transporters are classically classified as either “Uptake 1” (low capacity but high selectivity) or “Uptake 2” (high capacity but low selectivity) on the basis of their transport of neurotransmitters. Transporters with high selectivity for trace amines have not previously been described. We are therefore currently systematically investigating transporters classified as “Uptake 1” or “Uptake 2” on the basis of their substrate profile for the monoamine neurotransmitters, and examining for effects on trace amine release characteristics of selective inhibitors of each transporter in various brain regions. Transporters identified through this pharmacological screen will be investigated further following either the selective over-expression of the transporter or following selective knock-down of the transporter gene.

Future studies will also examine whether trace amine accumulation in peripheral cells is controlled by the same transporters as those identified in the brain. Since trace amines are present in a number of commonly ingested foods (red wine, chocolate, aged cheeses) the transporters involved in controlling the uptake of dietary trace amines in the GI tract will be examined in collaboration with Dr. Jane Alcorn.

In collaboration with Dr. Mark D. Turner we will shortly be beginning a project to determine the role of peripheral TAAR1 in controlling glucose and fatty acid homeostasis. This will initially use a combination of cell culture, selective gene knock-down techniques, and highly selective proprietary TAAR1 ligands developed by Hoffmann La-Roche and kindly provided by Dr. Marius Hoener. These compounds have been reported by others to prevent drug-induced obesity, although the mechanism of such effects are unknown. It has also been reported that the TAAR1 agonist 3-iodothyronamine can alter glucose and fat utilization, although the role of TAAR1 in those effects was not confirmed and 3-iodothyronamine is rather promiscuous.

Finally we have plans to collaborate with Dr. Janet Koprivnikar to determine the TAAR expression profile in various amphibian species. Aquatic vertebrates show a remarkable expansion of TAAR isoforms compared to terrestrial species. We hypothesize that this reflects the different thermodynamic properties required of signalling molecules for between individuals communication in a water versus air environment. Some amphibians have distinct aquatic and terrestrial life stages, whereas others remain aquatic. We hypothesize that the TAAR expression profile will vary as a function of terrestrial versus aquatic life stage. In the tadpole stage, innate avoidance behaviour is exhibited towards individuals infected with parasites which resembles the behaviour shown in the presence of predators. The chemical basis for this behaviour is unknown. Having determined the TAAR expression profile, we plan to determine whether TAAR play a role in the avoidance of predators and/or infected individuals. The role of trace amines and TAAR in predator-induced stress responses in mammals is also an area of possible study in collaboration with Dr. Jacqueline Blundell.

Prevention of cancer chemotherapy-induced toxicity

Cyclophosphamide is used in the treatment of various cancers and also as an immuno-suppressant. It is a member of the general class of alkylating agents, forming cross-links with DNA and thereby initiating cell death cascades. Cyclophosphamide itself is an inactive pre-cursor that requires metabolic activation in the body in order for anti-cancer, immuno-suppressant and toxicity to be observed. This metabolism occurs by the cytochrome P450 drug-metabolizing system. The toxicity profile of cyclophosphamide is well established and includes alopecia (hair loss), fatigue, nausea and hemorrhagic cystitis.

Prevention of alopecia

Alopecia is reported to be one of the most traumatic side effects of cancer treatment, in particular for women and children. Indeed, breast cancer patients have reported that induction of alopecia is more traumatic than potential mastectomy. Induction of alopecia by cyclophosphamide has been established to occur due to the induction of cell death in hair follicles. This cell death is dependent on the presence of a protein called p53. Interestingly, the majority of human cancers are associated with deactivating mutations in the p53 protein. A number of compounds have been reported in the literature to be able to prevent p53-dependent cell death. We therefore hypothesized that these compounds may prevent cell death in cells with normal p53 (such as hair follicle cells) while not affecting the cell death induced in cancer cells that have mutant p53. Such an effect may allow the toxicity of some cancer treatments to be decreased while not affecting the cancer treatment.

Cyclophosphamide-induced cognitive dysfunction

Increased anxiety and depressive disorders have been reported in patients undergoing cancer chemotherapy. While some of this may be secondary to the diagnosis of cancer, or onset of side effects such as hair loss, there is growing evidence that some cancer treatments themselves may increase the susceptibility to development of anxiety, depressive and other cognitive dysfunction. Such effects may persist long after the completion of chemotherapy. This decreased psychological well-being is a major complication for satisfactory patient outcomes, can cause decreased treatment compliance, and also causes a disproportionate burden on social and healthcare systems.

Chlorinated fatty acids

As part of the DNA alkylation mechanism of cyclophosphamide the two chlorine atoms are sequentially released as either chloride ions or chlorine radicals. These are highly reactive. Cells contain an enzyme called myeloperoxidase that uses free chlorine ions/radicals to form hypochlorous acid. This hypochlorous acid can then react with membrane lipids to release component fatty acids in the form of chlorinated fatty acids. Such reactions are thought to be involved in cell death and inflammatory responses. Given the abundant release of free chloride ions/radicals following cyclophosphamide administration, we hypothesized that this would yield an increase in the production of chlorinated fatty acids, events that may be up-stream of p53 activation in cell death cascades.

Cyclophosphamide work in the Berry lab

Our previous work identified a compound with a particularly intriguing profile of cell death response in cell culture models of chemotherapy-induced cell death in normal and cancerous cells. Our preliminary studies indicate that this compound can prevent cyclophosphamide induced hair loss in a clinically relevant in vivo model. We are currently determining the dose- and time-course of this prevention of toxicity.

We are also interested to see whether cyclophosphamide induces behavioural changes indicative of increased anxiety, depressive disorders or cognitive decline, and if so whether this occurs at the same doses as those which induce alopecia, and whether the changes can be prevented. In collaboration with Dr. Jacqueline Blundell we are determining the dose- and time-response relationship for cyclophosphamide induced changes in classical behavioural neuroscience models of anxiety, cognition and locomotion. Based on previously obtained preliminary data, we are also examining the dose- and time-dependent effect of cyclophosphamide on monoamine oxidase (MAO) activity in various brain regions. As one of the major degradative enzymes of monoamine neurotransmitters, changes in MAO activity could provide a molecular mechanism for any behavioural effects observed. Should changes in MAO activity and/or behaviour occur, we will then examine whether these changes are prevented by our rescue compound in the same manner as the induction of alopecia.

In collaboration with Dr. David Ford we are measuring the chlorinated fatty acid levels in various body tissues in the absence and presence of different doses of cyclophosphamide. Depending on results obtained future studies will examine the relationship between generation of chlorinated fatty acids and the initiation of alopecia, changes in behavioural activity, and changes in brain MAO activity. Whether chlorinated fatty acid production is prevented by our rescue compound, and if so over what dose- and time-range, will also be examined.