L18. Endocrine Toxicology

Question 1

What does endocrine mean?

Answer 1

Endocrine refers to the fact that the circulation is used as a means of communication throughout the body.

Question 2

What are the different endocrine systems?

Answer 2

1. Endocrine system
2. Nervous system
3. Immune system
   They are whole body systems

Question 3

What is the master gland of the endocrine system? Where is it located? What is an advantage of its location?

Answer 3

Central coordinator: pituitary. It receives input from the brain and re-input from the circulation.

• The pituitary sits out of the brain just below the brain.
• No BBB issues.
• Protein hormones can get to it.
• It sends out messages and the tissues it reaches send back messages.

Question 4

What is the sequence of endocrine system hormone release?

Answer 4

1. Hypothalamus
2. Anterior pituitary
3. Thyroid, ovaries, testes, adrenal glands

Question 5

What hormones does the hypothalamus secrete? Where do they go? What is their action?

Answer 5

• The hormones go through the portal circulation and affect the pituitary.
  TRH: positively stimulates TSH in anterior pituitary
  GnRH: Positively stimulates LH and FSH in anterior pituitary.
  Dopamine: suppresses prolactin (PRL) in anterior pituitary.
  CRH: Stimulates ACTH in anterior pituitary.

Question 6

What happens if you put the pituitary in the kidney capsule?

Answer 6

It doesn’t receive messages from the hypothalamus. Therefore the only hormone secreted by the pituitary is prolactin (PRL) because the pituitary won’t be inhibited from releasing it. All of the other pituitary hormones will not be released because they need to be stimulated by the hormones secreted by the hypothalamus. The only cells that have a break on them are the lactotrophs in the pituitary, all of the others need an accelerator.

Question 7

What are the hormones released from the anterior pituitary? Where do they go? What do they affect?

Answer 7

The hormones released from the anterior pituitary go into the central circulation and will affect different tissues.

• TSH stimulates thyroid to release T3/T4
• LH, FSH & PRL stimulate the ovaries to release estradiol and progesterone. they also stimulate the testes to release testosterone and estradiol.
• ACTH stimulates the adrenal glands to release corticosteroids.

Question 8

What happens when each of the tissues activated by the anterior pituitary hormones release their hormones/proteins?

Answer 8

The small molecules, like steroids or T3 or T4, will feed back to the pituitary and the hypothalamus. If they are proteins like inhibin that come out of the ovary or testis, it can only feedback at the level of the pituitary. Because it’s a protein hormone it doesn’t cross the blood-brain barrier.

Question 9

What is the main driver of the endocrine system? Give an example. What is this a target for?

Answer 9

• The main driver is negative feedback
• EX: LH stimulates testosterone and estradiol production. When estradiol and testosterone increase, the pituitary stops producing LH which means there’s less testosterone that will be produced. Then the pituitary will start making LH again to re-increase testosterone levels.
• Target for endocrine disruptors

Question 10

What is the steroid pyramid? How does this tie into endocrine disruptors?

Answer 10

1. Cholesterol is a steroid that is a precursor of all steroids.
2. It gets metabolized to progestins which is a family of steroids containing progesterone and pregnenolone.
3. The precursor to all androgens = progestins. Androgens = testosterone, dihydrotestosterone, etc.
4. You can’t get to estrogens without going through androgens. Estrogens = picograms/mL. The receptors respond to minute amounts of estrogen. Therefore if you have a contaminant in very low concentrations, it is more likely to take all the estrogen receptors than androgens progestins or cholesterol.

• Because of this pyramid, a lot of endocrine disrupters is related to the effects on estrogens and androgens.

Question 11

What is an endocrine disruptor?

Answer 11

• Any way you can disrupt the endocrine system.
• WHO definition: An endocrine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations.
• The tissues or organs cannot respond in an appropriate manner.

Question 12

What is the timeline for endocrine disruptors from 1930-1962?

Answer 12

• 1930-77: Widespread PCB (Polychlorinated bisphenols) use in transformers & as cutting oils
• 1942-72:Widespread DDT application in malaria control & agriculture
• 1941-54: FDA & USDA: DES approved for use in humans & animals
• 1959: DES produces cancer in experimental animals
• 1962: Publication of Silent Spring by Rachel Carson. The first of 2 books that had a major impact on the field of endocrine toxicology.

Question 13

What did Silent Spring by Rachel Carson show?

Answer 13

• The thickness in raptor shells is inversely related to the amount of DDT. More DDT= thinner raptor shells.
• The number of big birds decreased decade after decade.
• Then regulations came in and after DDT was banned in NA and the western world the numbers started to increase again.

Question 14

What is the timeline for endocrine disruptors from 1971-1996?

Answer 14

• 1971: The president of the company that made DDT said it was really safe and doesn’t need to be regulated.
• 1972: EPA bans DDT, FDA warnings on DES in pregnant women. DDT has a very long half-life and is still present in us. It is still used in some parts of the world.
• 1977: EPA bans PCBs
• 1979-1995: Meetings & publications on estrogens in the environment as potential endocrine disruptors.
• 1995: EPA held first federal meeting for endocrine disruptor workshop; NAS/NRC panel meets.
• 1996: Our Stolen Future, Colborn, Dumanoski & Myers, published; FQPA passed & Safe Drinking Water Act amended. The second book that came out that affected endocrine toxicology. The 3 authors of this book made a case that chemicals in the environment were having huge effects on public health.

Question 15

Who is Theo Colborn?

What was the book “our stolen future” about?

Answer 15

Theo colborn was A primary driver of the book “our stolen future” because the the authors were publishing the book in the 90s they received major threats from the chemical companies. And she spoke out about it in their defence.

The 3 authors of this book made a case that chemicals in the environment were having huge effects on public health.

Question 16

What is the timeline for endocrine disruptors from 1998-2015?

Answer 16

• 1998: International Conference on Endocrine Disruptors, Kyoto. National research council in US puts out their first volume.
• 1999: NRC report, Hormonally Active Agents in the Environment
• 2002 WHO Global Assessment of the State of the Science on Endocrine Disruptors. Saying there MAY be an issue.
• 2012: WHO Endocrine Disruptor Chemicals 2012. Very strong document.
  Very strong document.
• 2015: Endocrine Society takes position on Endocrine Disruptors. Clearly identifying what the endocrine disruptors are, what the families are, which tissues they act on. Now it’s a very respected field.

Question 17

Why does the publication by the WHO in 2012 on endocrine disruptors have an emphasis on pregnant women?

Answer 17

• Emphasis on pregnant women because during development, exposure to chemicals have greater impact on the children than on the adults.

Question 18

What are the families of chemicals defined as endocrine disruptors (EDCs) in everyday environment by the WHO?

Answer 18

• Polycyclic Aromatic Hydrocarbons
• Bisphenol A
• Cleaning Products
• Plasticizers/pthalates:
  Anything that will make a plastic more pliable. “New car smell” = inhaling plasticizers, makes lipstick glossy.
• Air pollution
• Air particulates
• Ozone
• Flame retardants
• Metals, Plastics
• Pharmaceuticals
• Pesticides
• Herbicides
• Fungicides

Question 19

How do EDCs get into the environment?

Answer 19

Via point and diffuse sources:

• Chemical company makes products that can go in the air, can go in products, can go in water, runoff into rivers, oceans, agriculture
• The foods we buy and its wrapping materials.
• They’re everywhere around us. The issue is not if we have them around us, the question is how much is there and when will it be a worry? How do we draw that line?

Question 20

What are the different actions of endocrine disruptors?

Answer 20

• mimic a natural hormone. For example: smt that binds to estrogen receptor and works like estradiol
• block the effects of a hormone
  Ex: binds to a receptor but acts an an antagonist
• directly stimulate or inhibit the endocrine system
  Ex: increase TSH or LH secretion which can alter feedback routes
• cause overproduction or underproduction of hormones
  Ex: if you have a chemical that inhibits conversion of progestins to androgens, it’s not working through the receptors but it will suppress the production of androgens and estrogens as a consequence. If you have a chemical that stimulates the transport of cholesterol into the mitochondria, you will increase stearin production.

Question 21

What are the endocrine targets?

Answer 21

• The endocrine target tissues are not simply the endocrine tissues from the endocrine system.
• Every tissues in the body has receptors for endocrine hormones.
• Skin, fat lipid cells can metabolize androgens to estrogens, bone (has receptors for androgens & estrogens), brain has receptors for androgens and estrogens and can also metabolize and produce some steroids. Kidney, lung, heart, all have receptors for endocrine signals.
• They may not all be part of the endocrine system but they respond to endocrine signals. So if you alter the endocrine signals, you can affect every part of the body.

Question 22

Why is pregnancy important in EDCs?

Answer 22

• Pregnancy is very important.
• From the formation of the gametes (sperm and egg) you can alter their composition so you can alter the development of the embryo and then the fetus.

Question 23

What is Dohad? What is it based on?

Answer 23

• The developmental origins of health and disease.
• DOHaD is based on evidence that the roots of many diseases and dysfunctions occur very early in life, especially the embryo, fetus, infant, and child
• Under- or over-nutrition of a pregnant woman or man has an influence on the fetus’s propensity to develop metabolic disorders including obesity, cancer, and diabetes later in life.
  Ex: Children of mothers who were starved in WW2 had altered metabolism. They had much higher incidence of diabetes, cardiovascular disease, and cancer.
• Developing germ cells are vulnerable to disruptions from even low doses of EDCs.
• The nervous system has been found to be very sensitive to EDC exposures.
• Some cancers, e.g., testis cancer, have their origins in early life.

Question 24

What is the idea of exposure to EDCs?

Answer 24

The effects of early exposures to EDCs

may be manifested any time in life.

Question 25

What are the Epigenetic Mechanisms of Endocrine Disruptors?

Answer 25

• The exposure to the EDC, modifies the DNA methylation pattern.
• The altered methylation pattern, when it is expressed later in life, results in the changes we see in the adults.

Question 26

What are some known EDCs and their uses?

Answer 26

• Pesticides: DDT, chlorpyrifos, atrazine (Has just been pulled off the market bc it affects the estrogen system).
• Children’s products: Lead, phthalates (toys and baby bottles), cadmium
• Food contact materials: BPA (white lining of cans prevents rusting but it gets into the food), phthalates, phenol
• Electronics and Building materials: Brominated flame retardants, PCBs Phthalates, organophosphate esters
• Personal care products medical tubing: Phthalates
• Pharmaceuticals: Contraceptives, drugs
• Antibacterials: Triclosan
• Textiles, clothing: Perfluorochemicals

Question 27

What are the endocrine disruptors in wildlife?

Answer 27

• Eggshell thinning in raptors – DDT
• Beak, skeletal, reproductive abnormalities
from POCBs (bald eagles, cormorants, alligators)
• Intersex fish below sewage plants

Question 28

Who is Louis Gillet?

Answer 28

• Biologist in Florida that worked with alligators. He was asked to investigate a lake where the population of alligators was going down.

Question 29

What are the potential sites of endocrine disruption in alligators?

Answer 29

• Same loops and hormones as us
• Brain: releasing factors
• Pituitary: LH/FSH/GH
• Liver: enzymes
• Gonad: Oestrogens/androgens
• Blood: binding proteins

Question 30

What did Louis Gillet do?

Answer 30

• There was a lake that had decreasing alligator populations and there was another lake where that wasn’t happening (Woodruff and Apopka lakes).
• He looked at the males and females in the 2 lakes and measured dihydrotestosterone (active testosterone metabolite) as well as estradiol.
• He found that in the lake of dying alligators, the concentration of testosterone was lower, and the concentration of estradiol was higher.
• He was able to link it to a chemical company who had the runoff of their production into that lake. The company had to then treat the chemical before putting it in the lake and 5 years later the population of alligators went back up.

Question 31

Explain the case of Increased Intersex in Fish (minows) in the St Lawrence river.

Answer 31

• They collected fish (minnows) from different parts of the St- Lawrence.
• Fish can be either male or female. They looked at the proportion of minnows that were intersex.
• They found that the proportion of intersex fish went up dramatically as you pass by the places Montreal dumps its water.
• So Montreal started cleaning its water and they went back 10 years later to see if the situation improved. They couldn’t find any more minnows. The population had disappeared bc the sex ratio had been thrown off and they couldn’t reproduce anymore.

Question 32

What are the major families of endocrine disruptors?

Answer 32

• Bisphenol A
• Phthalates
• Flame retardants: brominated flame retardants and organophosphat flame retardants
• FFOA/PFAs

Question 33

What is the history of brominated flame retardants?

Answer 33

Brominated flame retardants were introduced in very high quantities in furniture, cellphones, televisions, etc. by laws in California. Since California is a big state, the companies put it in everything so that they could sell it to everyone and other places in the western world. Firefighters pushed to increase the amount of flame retardants so things wouldn’t get into flames fast, particularly in pyjamas and stuff. They found that firefighters were having detrimental effects from the flame retardants they were inhaling from the fires, The flame retardants that were added to clothes were retarding the clothes from flaming up by 5-7 seconds which wasn’t really having an impact. Later, the same firefighter groups fought to remove flame retardants.

Question 34

What are organophosphate flame retardants?

Answer 34

The brominated flame retardants were replaced by organophosphate esters. But, one of the underlying problems in all regulations is that if a chemical is banned, or highly regulated, the chemical that is used by the company to replace it, does not have to be show that it doesn’t cause the same harm as the first chemical. Ex: if you’re replacing chemical A with B, you don’t have to show that chemical B doesn’t cause the same harmful effect as chemical A.

Question 35

What are FFOA/PFAs?

Answer 35

In teflon… all the things that prevent staining of clothes and couches are sprayed with these chemicals. You don’t find teflon pans anymore because they’re being replaced with other finishes. The contamination with PFAS has not been fully examined.

Question 36

What is Bisphenol A used in? What was canada the first country to do?

Answer 36

• Used in hard plastic bottles, dental fillings, lining of cans, etc.
• Canada was the first country, in 2010, to say that BPA should not be used in baby bottles or in teethers or toys for babies. They didn’t ban BPA, but it said it shouldn’t be in those products, then the US and Europe followed.

Question 37

What was BPA first replaced by?

Answer 37

• They first replaced bisphenol A with bisphenol S.

- There are over 28 bisphenol, very few of them have been tested systematically.

Question 38

How did they discover that BPA was a problem?

Answer 38

• A researcher moved to Washington state and found that her control animals had a higher level of aneuploidy than the rats that she used to be testing when she as in Ohio.
• For 2 years she tried to figure out why. She found out that in Ohio they were using different cages and bottles than in Washington. In Ohio they were using polyethylene polypropylene cages. In Washington they were using a polymer made with bisphenol A.
• Small amounts of BPA would leak into the mice from the cages and bottles and she was able to prove that the aneuploidy (abnormal chromosome number) in the mice was caused by this.
• Nobody believed her.

Question 39

What are the Adverse effects due to exposure to BPA during development or in adulthood? (animal studies)

Answer 39

• Kidney: abnormal growth
• You see prosthetic hyperplasia and prostatitis (infection in the prostate)
• Effect on breast cancer cells.
• They cause a decrease in sperm count.

Question 40

What were the conclusions of the meta analysis to see the endpoints affected by BPA?

Answer 40

• They thought that the effect on reproduction was the worst but that was not the case. The most significant effects were cardiovascular effects and diabetes.

Question 41

What is the effect of BPA on estrogen receptors?

Answer 41

• BPA binds to estrogen receptor alpha or beta at concentrations of 10^-4 to 10 ^-5 molar. Estradiol is in the range of 10^-11 molar.
• ERRy and ERRBeta proteins do not activate gene expression in the nucleus. They bind to ERalpha or ERbeta, dimerize with them, and prevent translocation into the nucleus.
• ERRY binds bisphenol A at 10^-11 molar concentrations. – This is the first non-monotonic dose response curve that was identified in endocrine disruptors.

Question 42

Explain the mechanisms of nonmonotonic dose-response relationships (BPA). (go look at L18 S48)

Answer 42

• Very low doses of chemicals like BPA can have effects.
• If you look carefully enough and focus on the endpoint you’re looking at, you can decompose any non-Monotonic dose-response curve into 2 or more Monotonic dose response curves.

Question 43

What is the glossy surface on a receipt caused by?

Answer 43

The glossy surface on a receipt used to be BPA now its replaced with BPS. Cashiers have very high serum levels of BPS. BPS is about as toxic as BPA. Other bisphenols can be less toxic or more toxic. BPTMC is a lot more toxic. Health canada wants to regulate the whole family instead of going after one chemical at a time. That has never been done before.

Question 44

What is the “clarity project”?

Answer 44

There’s a “clarity project” funded by the NIHS with the FDA in the US. Huge study that lasted 3 years. Over 30 papers came out of the project. It is very controversial. If you use the classical endpoints (the regulatory guidelines in terms of what is normally assessed) the regulatory conclusion is that BPA is safe. In the US BPA is very poorly regulated. If you look at the individual researcher projects that are measuring more refined endpoints than in the regulatory guidelines, every one of the researchers found effects of BPA at low doses.

Question 45

What are phthalates? Where are they found?

Answer 45

• Plasticizers.
• Plastic is polyvinyl chloride which is a very rigid polymer. If you try to bend it it will snap. To make plastic pliable you need to add something that interferes with the rigid structure and allow pliability. PTHALATES!
• Found in gloves, paint, makeup, etc.

Question 46

What is the predominant phthalate in use?

Answer 46

The predominant phthalate in use is diethol hexol pthalates (DEHP).

Question 47

Who has the highest phthalate concentration in their blood and why? What do phthalates do?

Answer 47

• IV bags, tubes, neonatal care units are fed and have bags and tubes in them, they have the highest pthalate concentration in their circulation

Affects the male reproductive system:

• Altered sex hormones: LH, FSH, Testosterone, Estradiol, Inhibin
• Reduced anogenital distance: hormonal exposure during gestation. The anogenital distance in females is smaller than in males. The more you block androgen action in gestation, the smaller the anogenital distance.
• Hypospadius: The urethra does not open at the tip of the penis, it opens somewhere on the penis shaft
• Cryptorchidism: The testis does not descend properly into the scrotum. If you stay in the peritoneal cavity, you will block mitosis. Spermatogenesis requires a temperature lower than core. You will also increase testicular cancer if you stay cryptorchid.
• Reduced sperm production and mobility.

Question 48

What are the endpoints that are affected by phthalate esters?

Answer 48

1. Male reproduction: Leydig, Sertoli and germ cells
2. Female reproduction: folliculogenesis, oocytes, early menopause
3. Adrenal: steroid production
4. Fat cells metabolism (of steroids)
5. Respiratory system: Asthma
6. Cancer: Liver, breast (still debating)?
7. CNS: Child neurobehavioral development

Question 49

What is the mechanism of action of MEHP?

Answer 49

1. Affects leydig cells:
   this causes a decrease in testosterone and Insl3 levels. Insl3 is a protein produced by leydig cells that allows testicular descent into the scrotum. Therefore, MEHP affect on leydig cells causes Hypospadias and cryptorchidism.
2. Affects sertoli cells (Cells in seminiferous tubules that supports spermatogenesis):
   - It affects sertoli cells directly but also indirectly via the lowering of testosterone production in the leydig cells because testosterone usually stimulates the sertoli cells.
   - result: decreased germ cell proliferation. the final effects is multi-nucleated germ cells which cause testicular cancer. This is why there is an associated higher incidence of testicular cancer in men.

Question 50

What is an AOP and what is significant about the AOP for phthalates?

Answer 50

• AOP = adverse outcome pathway.
• AOP’s are to try and figure out how chemicals work. The AOP approach is good at identifying what we know and what we don’t know and help us focus.
• There could be an AOP for the different types of endpoints. For example, an AOP about phthalates designed for a male reproduction endpoint.
• AOP’s start with exposure and then define the molecular interaction, cellular effects, organ effects, and organism effects of the chemicals.
• The AOP for phthalates on male reproduction shows that we don’t know a lot about the molecular interactions. However, we know about the cellular, organ, and organism effects.

Question 51

When looking at how industries are shifting towards “safer” alternatives for chemicals, what is the question?

Answer 51

Is it a regrettable substitution or a responsible replacement?

Question 52

Describe how MEHP and other alternative plasticizers can be analyzed or tested to check for their safety.

Answer 52

1. A cytotoxicity cell assay using MA-10 cells (which are like leydig cells, can be used in vitro). In this assay, you’re looking at cytotoxicity to see if the cells are dying when exposed to different concentrations of the chemicals. From this test we saw that there was no cell death in any of the replacement plasticizers at significant concentrations other than MEHP. However this does not give the whole picture.
2. When doing assays on leydig cells looking at oxidative stress and mitochondrial function, it was would that oxidative stress increased in the leydig cells and at least 3 of the alternatives affected mitochondrial function.
3. You can look at the Effects of Plasticizers on Phenotypic Endpoints in C18-4, MA-10, TM4 and KGN cells. We’re looking at 4 different cell lines and the different chemicals and what happens to lipid droplets, mitochondria, lysosomes, nuclei…different cellular elements. You’re asking if these chemicals are all affecting different components. Most of them do affect cellular function (TXIB showed no toxicity) and if you look at transcripts you can see that gene expression is being affected by all of them.

Question 53

What is a ToxPi analysis? What can it be used for?

Answer 53

• it’s an analytical technique that can be used to rank the potency of chemicals.
• It is a pie chart with each pie slice representing a different endpoint (ex: oxidative stress, total mitochondria, cytotoxicity, etc.)
• The area of each slice of the pie chart represents the potency of each endpoint. The bigger the area, the more potent the chemical is on the endpoint.
• Using ToxPi analysis, You can take 4 different cell lines (ex: all from the reproductive system) and all the chemicals, and you can have a very quick assessment of which cell line responds most to which chemical.