Toxicological Profile of Ultrapure 2,2 ',3,4,4 ',5,5 '-Heptachlorbiphenyl (PCB 180) in Adult Rats

Peer reviewe

A study of possible environmental modulators of neostriatal dopaminergic synapses, with focus on dietary fat and polychlorinated biphenyl 153

Environmental substances like polychlorinated biphenyls and dietary fatty acids have been suspected to influence cognitive functions in humans, although much of their specific effects on synaptic functions are still unclear. There are many contradicting studies of the cognitive benefits of polyunsaturated fatty acids. In some studies it is emphasized that children with cognitive impairments such as ADHD, may have impaired fatty acid metabolism, implicating that the genetic status may be of importance. Ingestion of polychlorinated biphenyls have been associated with negative effects on cognitive processes, although the effects seems to be highly dependent on their molecular structure exposure time, duration, doses. In this thesis, the effect of dietary fat content and polychlorinated biphenyl 153 was investigated in neostriatal dopaminergic synapses and included measurements of related neurotransmitters like e.g glutamate and serotonin. We included both male and female rats of two different genotypes namely the Wistar Kyoto rats and the spontaneously hypertensive rats as an animal model of ADHD, to investigate if genetic and hormonal status may influence the effect of these compounds. Initially the dietary study involved two distinct diets, one with a low level of dietary fatty acids (5%, w/w) with the other one supplemented with omega-3 polyunsaturated fatty acids (equal amounts of EPA and DHA) containing a high level of fatty acids (21%, w/w). The omega-3 supplementation led to gender dependent improvement in behaviour in the male SHRs, and included reduced levels of reinforcer-controlled activity, impulsiveness and inattention, with no or opposite effects in the female SHRs. Further biochemical studies on the neostriatal dopaminergic system, involved in reinforcement of behaviour, confirmed that the male SHRs had a reduction of the dopamine content in concert with enhanced homovanillic acid and calculated turnover ratio of dopamine, all of which were absent in the female SHRs. To further investigate if the effects achieved by omega-3 supplementation (this time with 80% DHA and 20% EPA), could be specific for omega-3 fat, we included an extra diet composed of high levels of fatty acids (21%, w/w) containing mainly lard which is rich in both saturated (40%) and polyunsaturated fats (60%) but with low levels of omega-3. In this second round we discovered that in the male WKY rats, both the lard and omega-3 enriched diets gave reduced levels of dopamine, tyrosine hydroxylase and vesicular monoamine transporter-2, without changing the extracellular dopamine metabolite homovanillic acid in neostriatum. In contrast to the lard enriched diet, omega-3 enrichment induced an additional 2-fold increase in dopamine turnover ratio in the male WKYs, as well as a significant decrease in the levels of the dopamine transporter. Another finding was that the young male SHRs at p30 were hypodopaminergic compared to age and gender matched WKYs. In these male SHRs, the 40% lower dopamine turnover ratio was reversed 2-fold, to a similar extent by both lard and omega-3 enriched diets, without any significant effects on the protein levels. These findings show that dietary fatty acid composition may strongly influence the neostriatal dopaminergic system in a gender and genotype dependent way, with both type of fatty acids as well as the amounts influencing the synaptic responses. Polychlorinated biphenyls (PCBs) can be separated into ortho- and non orthosubstituted PCBs, where most of the ortho-substituted being neurotoxic and possibly interrupt cognitive functions. In this thesis we employed the ortho-substituted PCB 153, which is one of the most abundant PCB found in mammalian milk. Biochemical studies were performed on dopamine and serotonin neurotransmitters as well as amino acids in the neostriatum of both genders from the Wistar Kyoto (WKY) and the spontaneously hypertensive rat (SHR) genotypes. Exposure to PCB 153 led to increases in homovanillic acid and 5-hydroxyindoleacetic acid in all groups except the female SHRs, whereas levels of dopamine and serotonin neurotransmitters as well as amino acids were unchanged in all genotypes and genders. PCB-153 also induced a decrease in the neostriatal D5 receptor in both genders and genotypes, without changing the D1 receptor. In contrast, levels of the dopamine transporter were reduced in the male WKYs, together with an insignificant reduction of the mean in the male SHRs. In addition, a gender-specific decrease of the PSD-95 protein occurred in the PCB-exposed male rats. Levels of tyrosine hydroxylase and vesicular monoamine transporter-2 were unchanged in all animals examined. Therefore, postnatal PCB exposure had major effects on both dopamine and serotonin turnover as well as specific PCB-sensitive synaptic proteins. Differences occurred between the effects obtained in both genotypes, as well as between genders. Altogether, this set of studies shows that both PCB 153 and dietary fatty acids, environmental compounds suspected to influence cognitive functions, may modulate neostriatal dopaminergic synapses in distinct gender and genotype dependent ways

PCB i miljøet som mulig årsak til utvikling av ADHD : Målinger av reseptorbinding og aminosyrenivå i hjernen

ADHD (Attention-deficit hyperactivity disorder) kjennetegnes av manglende oppmerksomhet, impulsivitet og hyperaktivitet. Omlag 80% av forekomsten er genetisk betinget, mens de resterende 20% trolig skyldes miljømessige årsaker. Eksponering av rotter under utvikling med PCB (Polyklorerte bifenyler) har visst å gi lignende atferd som ses hos dyremodellen for ADHD (SHR-rotter) (Holene et al. 1998). Studier av ulykker og arbeidseksponering har vist at PCB også kan forstyrre kognitive funksjoner hos mennesker (Mariussen og Fonnum 2001). I denne oppgaven ble to rottemodeller for ADHD, SHR og ADD, sammenlignet med kontrollrotter med normal atferd (WKY og SD) for å finne nevrokjemiske parametere som kan kobles til ADHD-atferden. I tillegg ble PCB-eksponerte rotter sammenlignet med rottemodellene for ADHD. Dette ble gjort for å undersøke om PCB-eksponering kan være medvirkende i utviklingen av ADHD. Ved målinger av aminosyrekonsentrasjoner ble det funnet signifikant lavere aspartat-konsentrasjon og signifikant høyere glysin-konsentrasjon i hele hjerner fra SHR sammenlignet med WKY-rotter. Forskjellene ble ikke funnet i striatum. Det ble i tillegg vist at voksne SD-rotter eksponert for høye konsentrasjoner PCB180 (1000 mg/kg) hadde tilsvarende økning av glysin-konsentrasjon i hjernen, i forhold til sine kontroller. Bindingsstudier av nikotinreseptor-subenheten α4/β2 ble utført på høyre og venstre hjernehalvdel og viste signifikant lavere binding i høyre hjernehalvdel i forhold til venstre hos SHR-rottene. Det ble derimot ikke funnet signifikante forskjeller i α4/β2-nikotinreseptorsubenhet-binding i hjernehalvdelene hos WKY- og ADD-rotter. α4/β2-Nikotinreseptor-bindingen var signifikant lavere hos SHR-rotter sammenlignet med WKY og ADD, både i total hjerne og i hippocampus. Eksponering av voksne SD-rotter for PCB180 (1000, 100 og 3 mg PCB per kg) og eksponering av unge SHR- og WKY-rotter for PCB153 (9 mg PCB per kg) viste ingen signifikant effekt på α4/β2-subenhet-binding i hjernen. Det ble heller ikke funnet noen signifikante forskjeller i NMDA-reseptorbinding i hippocampus fra SHR- og WKY-rotter eksponert for PCB153 sammenlignet med kontroll (ueksponerte SHR- og WKY-rotter). Dopaminreseptor-subenhetene D1/D5 ble undersøkt i hjernehalvdeler fra voksne SD-rotter eksponert for ulike konsentrasjoner av PCB180, men det ble ikke funnet signifikante forskjeller sammenlignet med kontroll

Treatment groups and doses. Loading dose was administered on study days 0–5 and maintenance dose on study days 10, 17 and 24.

1<p>The target total dose of group 8 was 2000 mg/kg bw, but due to unexpected decrease in body weight (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104639#pone-0104639-g001" target="_blank">Fig. 1</a>.) the third loading dose was omitted for animal welfare reasons, and the rats received only the corn oil vehicle.</p

Sensitivity of endpoints showing significant dose-responses in males (left panel) and females (right panel).

<p>Adipose tissue PCB 180 concentration based CED values at CES 5% (shown in log scale) are ranked according to sensitivity in males. Endpoints are grouped into biochemical/molecular endpoints, neurobehavioral endpoints and apical/hematological endpoints. Key: endpoints with significant dose-responses in both genders: black bars; endpoints significant only in males: blue bars; endpoints significant only in females: red bars; endpoints existing only in one gender: yellow.</p

Margins of exposure (MoE = CED-L/human median PCB 180 concentration) for different endpoints of PCB 180 toxicity and different human cohorts. MoE values exceeding the WHO default uncertainty factor of 25 are bolded.

<p>Human PCB 180 tissue concentration data: median, range (ng/g lipid).</p>1<p>Placenta <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104639#pone.0104639-Virtanen1" target="_blank">[40]</a> (1997–2001), Denmark: 9.65, 2.03–25.2; Finland: 5.56, 1.44–14.92.</p>2<p>Mother's milk, 58 milk pools from 18 European countries <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104639#pone.0104639-EFSA1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104639#pone.0104639-VanLeeuwen1" target="_blank">[2]</a> (2001–2002): 45.8, 6.1–337.</p>3<p>Adipose tissue, general population, Finland <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104639#pone.0104639-Kiviranta2" target="_blank">[96]</a> (1997–1999): 94.9, 11.3–833.</p>4<p>Adipose tissue, Baltic Sea fishermen <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104639#pone.0104639-Kiviranta1" target="_blank">[45]</a> (1997–1999): 460, 190–1200.</p>5<p>Adipose tissue, Baltic Sea fisherman with highest exposure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104639#pone.0104639-Kiviranta1" target="_blank">[45]</a> (1997–1999): 120.</p

Significant dose-responses of PCB 180 based on adipose tissue concentration.

a<p>Not available.</p>b<p>Calculated as the percent difference between controls and high dose according to the fitted model.</p>c<p>Half min value added to zeros.</p>d<p>Enzyme induction data from Roos <i>et al.</i>, 2011.</p

Sensitivity differences between males and females for endpoints showing significant dose-responses.

<p>Fold sensitivity difference is shown as the ratio of adipose tissue PCB 180 concentration based CED values at CES 5%.</p

BMD analysis of serum free T4 (A), serum free T3 (B) and serum TSH (C) in males (triangles) and females (circles).

<p>T4 and T3 were dose-dependently decreased in both genders, and TSH increased in males only. Small symbols indicate individual samples, large symbols the group mean; the vertical dotted line indicates the dose (CED) with 5% decrease or increase (CES -0.05) compared to background (a parameter). (Optimal models used for CED calculations as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104639#pone-0104639-t002" target="_blank">Table 2</a> were determined separately for females and males and are not necessarily the same shown here).</p