Dioxins 1993 talk by Linda Birnbaum.

[Ralph Ryder <ralph@tcpub.demon.co.uk>]

Dear Friends
        I posted the following piece on the science of dioxin at the
time of the EPA reassessment to some close friends on the net. A number
of them advised me to post it to the full list . It could well have been
posted in the earlier days of the web but I havent seem it. 
It is a brilliant talk by Linda Birbaum given in 1993 to the 102nd
meeting of the Great Lakes Water Quality Board in Chigago. Many of the
'old hands' in the debate will already have seen it, but a great many
newcomers will find it a fasinating insight into the complexity of the
science and mechanism of dioxin. I hope it provides useful background
information and show those communities theatend with applications for
waste to energy incinerators, especially in Britian and Europe** that
our countries scientists are lying when they say "these plants poise no
threat to the health of communities living near them, including the
developing foetus and nursing children." They cannot, hand on heart say
this, but hand on wallet they can!

**(Sorry folks, but I'm against being part of Germany's 'United States
of Europe' and  having 'unelected' folks dictate to me) 

Enjoy Dr Birnbaum's talk!


RE-EVALUATION OF DIOXIN
A Presentation by 
LINDA BIRNBAUM
Director, Environmental Toxicology Division U.S. Environmental 
Protection Agency. 
To The 102nd Meeting of the 
Great Lakes Water Quality Board.
Chicago, Illinois, July 15 1993.

Introduction.
        What I would like to do today is give you an overview of the
state of science of dioxin and health effects.  What I am going to talk
about first is the effect of dioxin itself. Later on I will return to
the issue that dioxin is but one of a family of chemicals and, if you
really want to understand the risk out there, we need to look at the
aggregate of all of them together.  But the important thing again is
that you have a planar molecule with halogens off to the side. I would
like to stress that most of the information we have has to do with the
chlorinated molecule, but the brominated molecules are just about as
equally toxic, and we know almost nothing about them, and almost no
environmental monitoring has been done to determine whether they are
really out there or not.

        Some properties of dioxin are relevant to its persistence and
its bioaccumulation in the environment and why we have a problem with
it. It is insoluble and is highly bioaccumulated in food chains. It's
very insolubility is a real issue from a research and regulatory
standpoint when the standard procedure concerns how much is in the
water, whereas in fact there is almost none of it in the water itself.
There is a very low vapour pressure which means that it does not
volatilise very much.

Exposure Pathways
        How are people exposed to dioxin? Food is the major source,
except for some major industrial accidents where there might be some
inhalation of dioxin or some skin absorption. Essentially greater than
95% of our exposure is by the food we eat, primarily meat, fish and
dairy products. Now a lot of the attention has focused on the
concentrations of dioxin in fish, so that may be higher than the
concentrations in meat and dairy products. But in the American diet, at
least up until the present time, most Americans do not eat as much fish
as they do dairy product. So we have approximately equal amounts of
exposure from fish as from dairy products. There may be susceptible sub-
populations who do eat a large amount of fish who will get more than the
average exposure to dioxin.

Sources.
        Dioxin gets into the food chain by bioaccumulating in organisms
in the food chain. How does it get into the food chain environment? Some
dioxins are directly eliminated into water, for example, from pulp and
paper mills. Most dioxin is released directly to the atmosphere and is
subsequently distributed worldwide through atmospheric transport.
Dioxin is very sticky, it binds to particles, is picked up on dust
particles in the winds and is blown around.  Nowhere in the world today
is free from dioxin. This is a worldwide contaminant and can be found,
with sensitive analytical techniques, even in the most remote places on
earth.

Eskimos
        When was dioxin first found in the environment, and when did it
start to accumulate? Some chemical companies have been trying to
convince us for a long time that dioxin has been around since the
beginning of time, and that it is a product of forest fires and volcanic
activity. Prior to the onset of heavy uses of chlorinated organics in
industry, which really commenced about 1930, levels were extremely low,
based on analysis of sediment samples. People have done analyses of
Egyptian mummies from more than 2,000 years ago and frozen Eskimos from
northern Canada and the levels are below the detection limit. Dioxin is
a product of the modern industrialisation. Other than forest fires and
volcanos, what are the other major sources? I think it is a point to
remember that, unlike polychlorinated biphenyls (PCBs), dioxins and
dibenzofurans have no known industrial use and they were never made for
any purpose. They are contaminants of industrial processes involving
certain organic compounds and chlorine. They are produced by low
temperature combustion at between 300o to 400o degrees centigrade. To
destroy dioxins, you have to go to over 1200o centigrade. But you can
form them at combustion temperatures characteristic of wood burning in
wood-burning stoves. In addition, dioxin is a product formed by the
chlorine bleaching of paper and pulp products. The paper industry has
been very responsible in decreasing their use of chlorine bleach, and
thereby tremendously decreasing their input of dioxin and dibenzofurans
into the environment. Of course, that is after 50 years of heavily
contaminating the sediments, which give us a long-lasting problem.

Major Sources
        What are the other sources of dioxins?  In the U.S.
Environmental Protection Agency (USEPA) dioxin reassessment, there was
an attempt to use total mass balance equations to determine where the
dioxin is coming into the environment.  A number of environmental
monitoring studies indicate that the levels do not appear to have
declined since the mid-to-late 1980s. This suggests that we still have
inputs as well as outputs, and that we are in a pseudo steady state. In
the Great Lakes, there was a peak of dioxins in the early '70s and since
then levels came down until about 1987. In the U.S., the major new
sources of dioxin appears to be hospital incineration. In Germany it
appears to be municipal waste incineration. It is not a major source
here because we use landfills but, if we switched to municipal
incineration, it may become a major source in the U.S. If anyone knows
about hospital waste incineration, they are totally unregulated and they
burn at very low temperatures, they have tremendous amounts of
chlorinated plastic, and therefore lots of dioxin are potentially
emitted from them. Diesel exhaust also appears to be a source of dioxin.
Metal smelting and refining appears to be another source, but we really
don't have a good mass balance. In summary, we can account for about 50%
of the new input into the environment, and we are uncertain where the
rest is coming from.

Hormones
        A typical toxic substance goes into the body and kills cells, or
does one particular thing. In contrast, dioxin does a lot of things and
should be considered as a hormone. I don't know what it is a hormone
for, in terms of the natural sense.  Hormones go into the body and they
have different effects on different tissues, they can have different
effect at different stages of development of the tissue, and they can
have different effects on different species. I think we can say the same
thing about dioxin. So you see a great deal of tissue specificity,
developmental stage specificity, or age specificity, or species
specificity in response.

Wasting 
        At very high doses, in all species we have looked at, dioxin
causes death. But, again, it is not your typical pesticide kind of
death, where you know you give a lot to the animal and it goes four legs
up in the air immediately. Death is usually preceded by a wasting
syndrome, the animals can easily lose 50% of their body weight before
they die and, depending on the animal, the time to death varies. So it
takes guinea pigs about two weeks, mice take about three weeks, so do
rats approximately, monkeys take about six weeks to die, for example. It
is an inexorable process. If the level of dioxin administered to the
animal, either acutely or following repeated exposure, once they reach a
certain body burden, it is like a switch is thrown and the animal will,
approximately x number of weeks later, die. We really don't know what
they die of. You can, for example, feed the animal and prevent the
wasting, and they still die. So it is not just because they used up
their body glycogen. The highest levels that we know that people were
exposed to, would have been lethal in guinea pigs, but they are, at
least in the order of magnitude or more, lower than the levels that
would kill almost any other mammalian species. So I think that the
reason we have not seen wasting or death in people is we may not have
bid high enough exposure, and that is not an experiment I think we are
going to try.

Enlargement
        At levels below that where you see wasting, you can see effects
on the lymphoid tissues and you actually have loss of the thymus and
spleen and at slightly lower levels, and in the adult male you can have
atrophy of the testis. But, again, these are all relatively high dose
effects.  Effects on the liver--there are some differences in different
species but, in general, you see enlargement of the liver, you see
accumulation of fat in the liver.  In some tissue, you have hyperplasia,
which is a proliferation of cells. The tissue actually gets bigger from
having more cells, and this occurs in the lining of the gastro-
intestinal tract, it occurs in the lining of the urinary tract, and it
occurs in the bile duct, which comes from the liver.

        In other kinds of cells, instead of getting hyperplasia, which
is an inappropriate proliferation of cells, you get squamous metaplasia,
which is an inappropriate differentiation of cells. It is not that an
eye turns into an ear, but in fact one type of cell turns into another
type of cell. It starts behaving like that cell. So, one of the classic
kinds of symptomatology that we see, not only in monkeys for example,
but, also in humans, is metaplasia of the meibomian glands of the eye.
Now, meibomian glands are little glands at the base of your eyelid that
secrete very small amounts of fluid. With exposure to dioxins, these
actually change and start producing waxy exudates on your eye that makes
vision very difficult and this is a complaint of people who have had
very high levels of dioxin exposure. They also complain of problems with
hearing. There are glands that line your ear canal which are call the
ceruminous glands and these also undergo an inappropriate
differentiation and start producing earwax. These are not the normal
glands that produce normal earwax. It might be possible to monitor
exposed people by collecting some of their earwax and measuring the
concentrations of dioxin and related chemicals in the earwax. It will be
a little hard to do mass balance equation to calculate their whole body
burden, but it might be an accessible source of tissue where you could
find out whether there had been exposure.

Chloracne
        Chloracne has been the hallmark of dioxin toxicity. This is not
the ordinary teenage acne but a very, severe persistent form of cystic
acne. People who had been exposed over 40 years ago to dioxins in
industrial accidents are still having active chloracne, not limited just
to their face and back, but all over their bodies. It is characterised
by hyperplasia, which is a proliferation of cells, by hyperkeratosis,
which is an altered differentiation of the cells, and by changed
pigmentation.  It is a very severe condition. You could say it is just
acne, but obviously it has changed the lives of these people.

Teratogen.
        Dioxin is a potent teratogen in a number of species in that it
causes actual gross structural abnormalities, cleft pallet, and
hydronephrosis in mice. In other species, it causes much more subtle
developmental effects. Effects that, until the last couple of years, we
were not really aware of, because they are the kind of effects that you
do not see in a standard teratology study. In a standard teratology
study, you dose animals during organogenesis, which is the major period
of differentiation of the different organs and tissues, and then you
sacrifice them just before they would normally be born. The
developmental effects we are studying now in fact are things that you do
not see until the animal reaches puberty, and then there are alterations
in the sexual functions and reproductive behaviour of the animals. So,
if you kill the animals at birth, you are not going to see what is going
to happen.

Hypersensitive 
        I will talk a little bit more about cancer. Dioxin appears to be
a carcinogen in fish, rodents and other mammals, including humans. But
dioxin can also modulate the immune system resulting in an inability to
fight disease. It is a very powerful immunosuppressant. It can also
upregulate the immune system so that you start becoming hypersensitive,
start developing autoimmunity and allergies. Depending upon the stage of
the animal and the species, sometimes you observe immunosuppression and
in other cases you observe upregulation.


Biochemical Effects
        Dioxin causes a wide variety of changes in enzyme levels and
causes biochemical effects. There has been some discussion about whether
these changes represent adverse effects or just biological responses. I
think many of them can be considered as biomarkers of the potential for
other effects to happen. These changes in enzyme levels, including
increases and decreases in the synthesis, leads to alteration in
metabolism of both endogenous and foreign compounds. For example, it
affects the way we handle glucose metabolism, induces cytochrome P4501A1
and P45OlA2 and regulation of other kinds of enzymatic; activity.

Estrogen, Androgens
        Dioxin modulates many hormone systems and their receptors. It
affects thyroid, gastric hormones and melatonin, which is a product of
the pineal gland involved in the circadian rhythm. It affects estrogen,
androgens, glucocorticoids and insulin. Dioxin causes the modulation of
growth factors and their receptors including, for example, Vitamin A. By
affecting hormone systems, you alter the homeostasis of the animal and
switch how the animal behaves. For example, dioxin can act both as an
anti-estrogen by blocking estrogen activity in the breast or the uterus,
and in other tissues it may act more like an estrogen. Dioxin causes
decreases in circulating thyroid hormones in, for example, rats, but it
causes increasing levels of circulating thyroid hormones in mice. I
think we have to be very careful when we talk about these effects to
realise again that hormones are very carefully modulated, and the way
that we maintain homeostasis in our body is by having a balanced level.
So, if you perturb the level, either by increasing it or decreasing it,
you are putting yourself at risk for problems.

Dysregulator 
        Many different molecules are involved in controlling the
activity of cells. But there are growth factors, such as epidermal
growth factor, which tells cells to divide, and there are factors like
transforming growth factor beta that tells cells to stop dividing.
There are growth factors like insulin growth factor, which is involved
in glucose metabolism and in many other functions in the body. Dioxin
modulates the activity of many of these, leading to altered growth and
differentiation.  Dioxin is therefore a very potent growth dysregulator.

Mechanism of Action
        How does dioxin cause these biological effects? One thing I want
to impress you with is that the mechanism of action is much more
complicated than we thought. A couple of years ago, we thought that TCDD
came in and bound to a protein called the Ah receptor and that went on
into the nucleus and interacted with DNA to lead to changes in the
expression of genes and of protein. We know now that it is much more
complicated. The way that the Ah receptor functions. it is never present
as an isolated protein. Its action is controlled by other proteins, so
that the Ah receptor binds to two molecules of the, proteins called
heat-shock proteins. Heat-shock proteins were first identified by
raising the temperature of an animal, and observing changes in these
kinds of proteins. These proteins control the ability of the receptor to
bind dioxins. Then there is another protein here, which has been
identified only by its molecular weight. I am going to come back to this
later. We call it p5o, but we really don't know what it does. We have an
hypothesis but no tests. We have this complex of four proteins binding
dioxin, and then something happens. I leave this purposely vague, other
than to say we lose the heat-shock proteins, we lose p50, but gain
another protein which, at this point in time, is a protein called arnt.
This complex -- and we don't know whether this forms in the cytoplasm or
in the nucleus -- functions in the nucleus, so that the complex that
actually alters genetic activity is composed of two proteins, as well as
TCDD. Then the dioxin can be recycled back. Heat-shock proteins are two
molecules and this is really a family of proteins, and they are
developmentally regulated. You have the possibility that, at different
stages, these may function differently in controlling the ability of the
receptor to bind the ligand.
        There is the possibility that arnt, the second protein which is
actually involved with the DNA binding, is part of a family of proteins.
TCDD bound to a receptor can interact with arnt 1, arnt 2, arnt 3, arnt
N to form this heterodimer, the two protein complex. Each of these will
only recognise one specific gene. So we have multiple levels of
interaction going on here. We have multiple transcription factors,
multiple DNA recognition sites, and we also know that even the Ah
receptor, which at one point in time we thought was the product of a
single gene, has multiple alleles of that gene so that there are Ah
receptors that are slightly different sizes. We do not know yet about
the specificity of the receptors and whether the receptor for the
protein with a size of 106 kilodaltons will accepted the 97 kilodalton
protein, in addition there is a 110 kilodalton receptor. We do not know
whether or not these have any functional implications but at least there
is that possibility. The point is we are not dealing with a simple
system. This is really quite complicated.

Alters Transcription 
        To make it a little more complex, Figure 2 is a hypothetical
schematic of the two ways that the dioxin Ah receptor complex functions
to alter the genetic activity of the cell. First, it changes it so that
you have more protein synthesis or less protein synthesis. And this is
what has been demonstrated and what people have spent most of their time
looking at.  That, in fact, TCDD binds with this complex, which then
falls apart and the dioxin receptor, plus that arnt protein, goes into
the nucleus, and alters transcription and gene expression. Second, this
p5O, which I told you about and we really don't know what it is, there
is at least an hypothesis that this protein is a tyrosine kinase. These
are proteins that put phosphate groups on tyrosine residues.  Tyrosine
is an amino acid in proteins, and when you phosphorylate proteins, you
affect their activity dramatically. With some recent studies from a
number of laboratories, including our own, we can show tyrosine kinase
activity is activated minutes after dioxin exposure. Things are
happening too quickly to involve changes in what is going on in the
nucleus. They involve changes that are going on at the point of contact.
We know also that some of the proteins that are phosphorylated by
tyrosine kinase in response to dioxin are proteins that control the
movement of cells through the cell cycle.  Here we have a direct tie-in
between a very rapid effect of dioxin, which clearly requires the
receptor because it acts as a regulator of this protein. When this
protein is bound to the receptor, it can't phosphorylate other protein.
When you release it from activity, it now goes on and does its thing.
One of the critical differences about dioxin as compared to, for
example, compounds in broccoli, cauliflower, and other foods we eat that
also bind to this receptor, is that those compounds are very rapidly
metabolised and gotten rid of. So you have a very short-lasting effect.
The problem with dioxin is that you have a persistent effect. Dioxin
comes in, it binds its receptor, and it ties it up. So you get a
constant signal from the nucleus to make more protein, and you have a
constant phosphorylation going on. Usually phosphorylation events are
very tightly regulated. They are usually very much one-off and now you
locked the switch. I think the whole idea of the persistence of the
dioxin activity is an important one to think about when we try and
understand what the compound is doing.

Amplification
        So what I have shown you is that this binding to the Ah receptor
is necessary for the effects of dioxin, it is the first step, but it is
clearly not sufficient. Many other things have to happen subsequent to
binding to the receptor. I think what you really have is a biological
amplification of these responses and it occurs by a cascade of growth
factors and hormones and the term that is used in the literature now
when we talk about steroid hormones, the estrogens, the gluco-
corticoids, the progesterones, is that you have "combinatorial
complexity." What that means is that you have a complicated network --
nothing is linear -- everything is interacting, and that is exactly what
is happening with dioxin. It is almost like dioxin is going into the
middle of this network, and sending out signals in all directions.

Toxicity
        There is a wide variation in acute toxicity of TCDD to adult
mammals. The guinea pig dies a week or so after being exposed to dioxin,
whereas hamsters survive until you get to very high doses. You have
approximately 3-5,000 fold differences in sensitivity. For many
chemicals this is highly unusual. For something that functions like a
hormone, this is not totally unexpected. But the point that I want to
gather is that, while guinea pigs are exquisitely sensitive, and I am
going to stress that these are adult guinea pigs, and adult hamsters are
very resistant, most mammals tend to cluster in the neighbourhood of 100
micrograms per kilogram as approximately the lethal dose. So while the
guinea pigs and the hamsters are outlyers for this response, most
animals are similar. I stress the point about adult hamsters versus
adult guinea pigs because if you look at hamster embryos or fetuses,
they are essentially equisensitive to guinea pig embryos. If you look at
rat embryos, they are essential equisensitive. There is something about
the adult hamster that makes them resistant to TCDD, but the embryo
responds at the same concentration as lots of other species.

Reaction 
        With respect to dioxin, people react similarly to animal
responses. Biology is inherently conservative and things tend to work
the same way in many species.  There is a large amount of data showing,
for example, that changes in biochemical properties such as enzyme
induction, in some hormonal states and in growth factors occur at
similar body burdens in animals as they do in people. For example, in
the ongoing occupational study conducted by National Institute of
Occupational Safety and Health (NIOSH) looking at workers who were
exposed to dioxin, these adult males are showing decreases in the levels
of their circulating testosterone at body burdens very similar to the
body burdens in adult rats. In immunotoxicity testing, human lymphocytes
and cultured cells respond to the same concentration of dioxin in the
media as mouse and monkey cells. in terms of developmental toxicity
based on organ culture you find similar responses at similar
concentrations of TCDD. For example, if you take out the embryonic
palate of a rat and the embryonic palate of a human, put them in culture
and expose them to the same concentration in the media, you get a
similar response.  Similarly, the body burden associated with chloracne
in people is essentially the same as the body burden causing chloracne
in monkeys, in hairless mice, or in rabbit cars. Animals with a lot of
hair -- like regular mice and regular rats -- do not develop chloracne,
but hairless mice do and the body burden there is essentially the same.
Cancer appears to occur at similar body burdens in animals as in humans.

Subtle
        I will just mention some really recent data on subtle
developmental effects that you would not see if you only looked when the
animals were born. Dick Peterson at the University of Wisconsin is doing
some very important mammalian work. A year ago he reported that if he
treated pregnant rat dams towards the end of gestation with a very low
level of dioxin, as low as 65 nanograms per kilogram, it resulted in de-
masculinisation and feminisation of the male offspring. Most of these
changes were not detectable until they reached puberty.  We have since
repeated that study, not only with his kinds of rats, but also in
another strain of rats. We have also looked at hamsters, and we get
basically the same result. We see decreased sperm count, altered sexual
behaviour, and shortened genitalia in these male rat pups.  We have
looked at both female rat and female hamster pups and we see even more
dramatic changes in the females, where we see hypospadias, which is
where the urethra, instead of emptying in a separate opening at the top
of the clitoris, actually empties into the vagina.  We see complete
clefting of the clitoris, and a cleft being maintained all the way down
to the vagina. In the rats, we see delayed vaginal opening and, in some
cases, no vaginal opening.  In the hamsters we cannot oven find an
external vagina in some cases. Clearly, the animals with no vaginal
opening are not going to be fertile. Although these animals appear to be
cycling perfectly normally and the ovarian-pituitary axis appears to be
functioning properly, we do not really understand the mechanism of what
is going on and we are trying to explore it.

PCBs
        These are very concerning events and Dr. Guo from Taiwan, who is
one of the principle investigators on the Yucheng cohort, visited my lab
two days ago. This is the PCB rice oil poisoning in Taiwan in 1979,
where about 2,000 people unfortunately cooked with rice oil that was
contaminated with PCBs that were themselves contaminated with the
polychlorinated dibenzofurans. The children born following this episode
have been followed for the past 8-13 years, that is the age of the kids
now. When they were first born they were reported to have what was
called ectodermal dysplasia syndrome, which included all sorts of
pigmentation problems, problems with their nails and dentition, and they
were small in stature. When they did development milestones, these kids
were developmentally delayed. They have continued to follow these kids.
Their IQ is shifted about five points down from the rest of the
population, and this has been maintained as they have grown up. It is
not something they have outgrown. The children continue to be shorter in
stature than matched controls and as the boys approach puberty, and some
of them are now between the ages of 8-13, the ones who are 10, 11, 12
and 13 are apparently having problems with their genitalia. This is very
new data, some of it will be presented this fall at the Dioxin Meeting,
but it is very compatible with the data that we are seeing in the
experiments.

Cancer
        Dioxin is a carcinogen. There are at least 18 studies in
mammals, all of which are positive. You may have heard that dioxin is a
tumour promoter, and not a carcinogen, because it does not directly
interact with the DNA. I think we start to dance on the heads of pins
because when I am saying dioxin is a carcinogen here, if you feed
animals in long-term studies without adding any known initiator, dioxin
by itself still causes an increase in tumours. It does not cause only
one type of tumour, it causes tumours at multiple sites. It causes it in
both males and females, and it has been detected in rats, mice and
hamsters. In addition, work from the U.S. EPA laboratory has indicated
that dioxin causes increases of tumours in medaka, at multiple sites and
short latency and at high incidence.

        There are three recent human epidemiology studies which, I
believe, deserve extra weight when we look at the dioxin epidemiology
literature. Prior to these studies, there were probably equal numbers of
studies that said, yes, dioxin does cause tumours in people and, no, it
doesn't. The advances that these three (Fingerhut et al., 1991, Manz et
al., 1991, and Zober et al., l990)* have are that they have blood levels
for the cohorts. So they can actually validate their exposure in their
industrial hygiene matrices with serum levels of dioxin. In that case
you find an increased standardised mortality ratio related to exposure
to dioxin, especially in the people who were exposed long term; people
with at least 20 years of exposure. This was a generalised tumour
response. I think you are all familiar with diethylstilbestrol (DES)
that specifically caused vaginal adenocarcinoma in young women. The
specificity of the lesion is why we were able to find out that this was
a problem. If you have something that causes a generalised increase in
cancer, it is very hard to pick up. There is a suggestion from two of
these studies that there may be an increase in lung tumours. Well, with
the background as high as it is in lung tumours, it is very hard to pick
up a small number of extra cases. But in fact these studies are all very
compatible with each other, showing that high levels of exposure to
dioxin are associated with an increase in cancers overall. There is
another study that will be published in the September issue of the
American Journal of Epidemiology, based on the Seveso cohort. Seveso was
a town in Italy, and in 1976 there was an explosion at a trichlorophenol
plant and the area around it was highly contaminated. The serum levels
in some of those people were the highest that we have ever seen. Until
now there has been a suggestion in a report published in 1989 that there
might be a increase in cancer, but it was just too soon, and the numbers
were too small. This paper in press now actually demonstrates, based on
cancer registry data in that area of Italy for 11 years since the
explosion, there are very significant increases in multiple types of
tumours in that population. Now that you think it is all bad, there is
also a decrease in breast cancer. Remember I told you dioxin is a
hormone, and it may increase some things and decrease other things. The
decrease in breast cancer, by the way, has been reported in animal
studies. In this report on the Seveso study to be published, the
increase is in both males and females, and again at multiple sites. I
really find it hard to accept the negative, or the null hypothesis. To
me, the data is overwhelming that dioxin has the potential, at least at
high doses, to result in cancer in people.

Dose-Response 
        If we look at the dose-response relationships for dioxin
interaction with the receptor will occur at the lowest concentration.
The activation of the receptor interacting with DNA may require
additional steps, and then all these other effects including enzyme
induction, immunotoxicity, developmental effects occur at much lower
levels than, for example, chloracne or cancer.  You have to have very
high levels of exposure to dioxin before you see chloracne. In cancer,
in order to detect an increase in tumours, you have to have even higher
doses again. With the immunotoxicity, one thing I should mention, is
that in the Taiwan cohort, the children, not the adults, are reported to
have elevated incidences, not only of respiratory infections, but also
otitis, ear infections.  In the northern Quebec Innuit population
exposed to very high levels of PCB relative to the general population,
children also have very high incidences of respiratory infections and
otitis, and also a very decreased rate of take of vaccinations. All
which would be at least compatible with the effects on the immune
system.

Endometriosis
        A recent report presented at the American Society of Gynaecology
has indicated that exposure to dioxin resulted in endometriosis in TCDD-
exposed rhesus monkeys, many years after the cessation of exposure.
These monkey were part of a cohort that was being studied at the
University of Wisconsin. Seven years after the termination of exposure,
one of the higher dose monkeys died after evincing severe pain and an
autopsy revealed that it had fulminating endometriosis. Since then, one
other monkey has died, and it died of the same cause. In monkeys
endometriosis can he fatal, though it is not fatal in humans.  There was
a Canadian study out of Health and Welfare Canada which had reported, in
abstract form, a suggestion of increased endometriosis in monkeys
exposed to Arochlor 1254, which is the PCB with the highest
concentration of dioxin-like PCBs in it. Because of all these findings,
the Endometriosis Association paid for some veterinarians and some
gynaecologists to do laproscoptic surgery on all the monkeys.  There
were controls, monkeys exposed to five, and monkeys that had been
exposed to 25 parts per trillion of dioxin in their diet and they bid
been exposed for four years, but that exposure had been terminated 10
years before this laproscoptic examination. There was a dose-related
increase in both the incidence and severity of endometriosis, as
compared to not only the control monkeys from this study, but to their
historic controls from their monkey colony, which involves something
like 300 monkeys. There are now at least two studies that are suggestive
of an association, or an increased incidence of endometriosis in these
monkeys and there is a possibility that it has fairly significant human
application.

Potency Rankings
        Dioxin is but one of a family of compounds including the
naphthalenes, the dibenzofurans, the biphenyls, both the azo- and
azoxybenzenes and then there were additional compounds. Remember,
naphthalenes were commercially produced between World War I and World
War 11, and the halowaxes were used to finish the nice wood flooring on
your ships and, in fact, there were numerous incidences of chloracne
occurring in some of the workers. These compounds are probably the major
actors though, that when they are halogenated in the lateral positions,
can interact with the Ah receptor and cause the same spectrum of
biological responses.
TEQ
        These chemicals all act by the same mechanism, and we should be
able to assign them relative potency rankings. The toxic equivalency
scheme is a scheme of relative potency ranking. These compounds are
considered as if they were a dilution of TCDD. So you weight them and if
you assign TCDD a value of 1, you can see that the brominated dioxin is
about 1/4th as toxic as TCDD itself. The tetrachlorodibenzofuran is only
1/20th as toxic despite looking like TCDD and binding very tightly to
the receptor. It is much less persistent than TCDD and is readily
metabolised in the mammalian systems, so the exposed animal can get rid
of it, since metabolism for these chemicals is a detoxification process.
The brominated-dibenzofuran is more toxic than the chlorinated, while
for the dioxin it is the reverse, because the brominated dibenzofuran is
harder to metabolise than the chlorinated. You can rank these compounds
(Table 8) according to their relative effect. This was done for cleft
palate, and it has been used in the development of toxic equivalency
factors (TEFs) and many other kinds of end points have also been used.
For example, receptor binding I have already mentioned. Induction of
biochemical responses, like enzyme induction, and this can be done
either in the animal in vivo or can be done in test tubes with cultured
cells, teratogenicity, effects on the skin, dermal toxicity,
immunotoxicity, or tumour promotion. All these things have been used,
and looked at in total to come up with a toxic equivalency scheme. The
U.S. EPA came out with interim TEFs in 1989, which I think is
essentially identical to the NATO values also today being used by
Scandinavian countries, and I think Canada uses the same numbers for the
dioxins and furans.

Metabolised
        The question is, what about the dioxin-like PCBs? The U.S. EPA
is determining values for TEFs for the dioxin-like PCBs in fish, we have
been looking at them in a long term mouse study, and Health and Welfare
Canada has been looking at them in a long-term rat study.  What we are
finding is similar, which is that while the dioxin-like PCBs must be
considered in any kind of assessment, in most cases they are not going
to drive the reassessment. Congener No. 77 binds the receptor very well,
but it is very rapidly metabolised in mammalian species and by many
fish. So its in vivo toxicity is much less than you would predict based
on something you did in culture. On the other hand, birds have very
limited ability to metabolise these compounds, so you want to assess the
toxicity for birds, using in vitro tests, while for species that can
metabolise it, you may want to use in vitro. In other words, TEFs have
to be applied with a certain degree of thought behind them.

Consensus Points
        So, at this point, I think in the general scientific community,
there are three consensus points. In general I think the scientific
community would say: i) that dioxins are growth dysregulators that are
mediated through the Ah receptor; ii) that people are sensitive to the
effects of dioxin; iii) and if you are going to look at the
environmental risk from these compounds, you need to consider all the
related isosteric compounds.

Risk Assessment
        There are two approaches you can take to estimate dioxin risks.
One is to use a biologically-based dose-response model, the other is to
take any mathematical model. We prefer to put more science into the risk
assessment process and develop models which are based on the mechanism
of action of the compound in extrapolating from animal to human data,
and from high to low dose. It is important to stress that there is no
evidence that all receptor-mediated responses must be non-linear. In
fact, there is no evidence that there is a threshold for responses such
as interaction with the receptor and simple biochemical events. Now that
says nothing about more complicated responses. I can't tell you yet
whether tumour promotion or immunotoxicity or developmental toxicity may
have thresholds or not. But I should stress that simple biochemical
responses are occurring in the same range of body burden as
developmental toxicity, and immunotoxicity. And in that range there is
no evidence for a threshold or non-linear response. The other point I
should make is that for those compounds we are not even starting from
zero exposure. We all have these compounds in our body.  There are lots
of these molecules floating around in us. so we are not starting from an
absence -- we are already somewhere up on the curve.
Similarities 
        So, one approach is the modelling and the extrapolation of the
experimental data. The other approach is to do some direct comparison of
body burdens with responses in animals and humans. What we see are great
similarities across species, and that there may be sensitive
subpopulations, based on either exposure or susceptibility. Examples are
subsistence fishermen who may eat much more fish and, therefore, have
higher exposures, or nursing infants, since the only way we know of to
reduce our body burdens from all these persistent lipophilic chemicals
is, if you are a woman, have a baby. That is not a very cheerful
thought, but what you are doing is partitioning the compounds out of
your fats into the milk fat and eliminating them by the milk.  During
the short period of nursing, the infant will be exposed to much higher
concentrations than for the rest of its life.

        And then there is the issue of susceptibility. In the animal
data, both the aquatic; data and the mammalian data suggest that the
embryo/foetus is the most sensitive stage for the toxicity of these
chemicals. We really don't know how sensitive the neonate is, who is
getting this very large exposure. In most of the mammalian studies that
I am more familiar with, the effects that are happening are occurring at
the end of gestation in rodents of at the early neonatal period.  But
that entire range of development will all be in utero in humans.

        Dioxin levels are normally expressed on a lipid or dosage basis.
Because of the persistence, there is an age-associated increase in the
levels. For TCDD, all of us sitting in this room have approximately 7
parts per trillion. If we look at the toxic equivalents for all the
dioxins and furans, it will be about 30 parts per trillion in our
bodies, and if we include the dioxin-like PCBS, that is going to be in
the neighbourhood of 50 parts per trillion. These are background levels
in humans in an industrial country such as the U.S. or Canada or Western
Europe and Scandinavia, eating food from the grocery store. Nobody has
looked at Eastern Europe and I bet there are areas where that is not
going to be true. I was going to remind you that, of the 209 PCB
congeners, only a very small number have dioxin-like activity, but the
non-dioxin-like congeners have their own inherent biological activity
and we would be mistaken if we ignored that, or if we thought that being
protective of dioxin-like PCBs would protect us against the non-dioxin-
like PCBS. Our current "dioxin" exposure is somewhere in the
neighbourhood of maybe 30-50 parts per trillion on a toxic equivalency
basis, derived primarily through the food. This kind of body burden of
dioxins and furans is associated with exposure to about 1-3 picograms
per kilogram per day. But nursing infants and subsistence fishermen may
have higher levels of exposure.

        So I think there are two views that can be looked at. The first
one is, are current levels in the environment a problem? The second is
that if we do not think they are a problem, should we worry about these
special populations, the nursing infants and the subsistence fishermen?
Do the current levels have the entire population on the brink of some
kind of biological response? I am purposely vague, I am not saying
adverse effect. Death is clearly an adverse effect, but is alteration of
your hormonal status an adverse effect? I am not sure. It probably
depends in what environment you find yourself. You know if your
glucocorticoid levels are already elevated and then you are stressed,
you might have a lot more problems than someone else. But are we at the
level of beginning to expect responses? In fact, are there people in the
population who are already experiencing subtle health effects? I should
mention that male sperm count has dropped over 50% in the last 50 years,
the incidence of endometriosis in the human population has increased
dramatically, the age of menarche has decreased dramatically and this
cannot all be accounted for by nutritional changes. I mean there are
definitely things going on there. There is a recent suggestion that
elevated levels of DDT are associated with increased incidences of
breast cancer. Are there subtle things going on? I don't really know the
answer to that but, if they are, then clearly, any increase of
individual exposure would be undesirable.

Dioxin Reassessment
        Now, before I tell you what I think, I just would like to
briefly mention the dioxin reassessment. About three years ago, there
was a meeting on dioxin at the Banbury Center in Connecticut and, when
we came back from it, I was really excited. I was also real naive. I had
just joined the agency, so I wrote Erich Bretthauer a memo telling him I
thought this was an opportunity for the agency to get in front of the
issue, instead of always coming in at the rear, and that there was
enough new information that had been gathered about the effects of
dioxin that we really ought to reassess its risk. In fact, Bill Reilly,
a little over two years ago, decided that we would do that and we
started a multi-faceted approach including a bioaccumulation project and
an aquatic toxicity project. There are many other parts of this
reassessment.  There has been evaluation of the literature, which has
been on-going. This is a critical review of the new literature.  There
have been eight chapters written, and they have been done by outside
experts in conjunction with U.S. EPA people as well. These were peer
reviewed last September at a meeting and have been undergoing re-
evaluation and updating really since that time. There is also analysis
of the exposure information. I should say there are actually eight to
nine revised chapters that will be available shortly, the exposure
scenario was also reviewed last September and that has been revised, and
there are three volumes of exposure assessment. We have been looking
more closely at human tissue levels, in collaboration with colleagues at
the Center for Disease Control (CDC). We have been doing a lot of data
collection, because what we wanted to do was try to develop
extrapolation models that would be biologically based. There was some
data that we felt we were missing, and we tried to identify the most
sensitive responses that we could measure and obtain better estimation
of the toxic equivalency factors for the coplanar PCBS. All these
different kinds of bench and laboratory science have been ongoing and
are feeding into a risk characterisation. We had originally hoped this
would be done by now, but we have been held up by the epidemiology
because all this new information which is just coming out, for example,
the paper in press about cancer in Seveso, needs to be incorporated into
the document. I think we are currently looking at public release of
drafts of these documents probably in about October, which means that
they would be taken to our external Science Advisory Board for review in
probably January or February.

        One thing we have tried to do in this reassessment is keep it a
very open process. We have done lots of public meetings. We have
solicited comments from the public. If anybody has additional comments,
we are more than welcome to entertain and listen to it and incorporate
it into the process. When we go out with the draft in approximately
October, we will also hold a series of additional public meetings,
probably in several regional offices across the country so that there
will be greater access, as opposed to holding those kinds of meetings in
Washington.

        So, what were the results of last September's review? Well, the
outside panel, at that point, said that the body burden in the general
population was at or near the level where responses are expected to
occur. I am going to editorialise, and I am not speaking for the agency,
I am only speaking for me, but this sounds to me that this has a direct
impact on a regulatory agenda. 
Thank you.


Questions:

David Villeneuve, Health and Welfare Canada: You have made reference to
neurobehavioral effects in connection with the Yucheng study, I believe,
and PCB and dibenzofurans. Is there any indication whatsoever that
dioxins are perhaps implicated in neurobehavioral effects?

Linda Birnbaum: Yes, there is. Prenatal exposure of monkeys appears to
result in altered spatial memory. You can tell monkeys that they are
supposed to remember where a certain object is, and the dioxin-exposed
monkeys have much more trouble with this short- term spatial memory than
animals who were not prenatally exposed. Most of the Yusho and Yucheng
data on responses is purely correlative, but if you try to associate
them with the level of PCBs or the level of dibenzofurans they correlate
with the dibenzofurans level much more than the PCB level for most of
those things. So we know that certain PCBs, like some of the non-dioxin-
like PCBs, are developmentally neurotoxic, but dioxin itself also
appears to have a different spectrum of developmental neurotoxicity.
Clearly, the sexual behaviour effects are neurotoxic effects, but they
were induced developmentally.

Gerald Rees, Ontario Ministry of the Environment and Energy: Would you
say that as a result of the dioxin reassessment we now have a better
understanding of the implications of dioxins or are there more questions
than when you started?

Linda Birnbaum: The answer is yes to both. Because if you do a good
scientific study, you always raise more questions than you answer. But I
think we have learned a tremendous amount. I personally feel that the
identity between animals and humans is much stronger than it was couple
of years ago. I mentioned the testosterone studies that have come out of
the NIOSH cohort. There are also indications that highly exposed
populations, not only the industrial workers but the small number of
ranch-handers from Vietnam who were highly exposed, they also had the
problems with testosterone and they also had problems with the glucose
tolerance test and an increase in diabetes, and so did the NIOSH cohort.
We don't know yet, and they are busy looking at that, whether it is Type
I or Type II, Type I being auto-immune and Type II being age-associated.
The ranch-handers also had increases in circulating immunoglobulin A
(IgA), which would change the immune system, and increases in
circulating lipids. Now those ranch-handers come back every five years
for a follow up. They were back in '92. It will probably take 2-3 years
before we have the results of that analysis. I think we are going to see
a lot more information out of the Seveso cohort in the next couple of
years. If we are going to do some more epidemiology studies, we need to
look at the right population, and I don't think the right population is
adult males. I think we need to be looking at adult females and we need
to be looking at children born to women who were exposed and we need to
follow those kids, especially for when they hit puberty.

Milton Clark, U.S. Environmental Protection Agency: One of the results
that is coming out now is about the criticism of U.S. EPA's conservatism
with dioxin relative to this non-threshold linear multi-stage model. Do
you feel fairly comfortable we were on the right track some years ago,
particularly when you look at the Fingerhut studies which show that when
you extrapolate from the workers studies it models pretty closely the
Kociba/Dow studies?

Linda Birnbaum: You are asking me a question I am not supposed to
answer.  Because until the reassessment is complete, we are not supposed
to say anything. But I will say if you do the linearised multi-stage
model, you come out with a certain number and if you now do a reference
dose model, but look at the neurobehavioral or developmental effects,
you come out with the same number, actually, even lower sometimes.

Milton Clark: But the Fingerhut extrapolation of the doses fall pretty
closely on the cancer incidence.
Linda Birnbaum: If you take the human data. Well, you can do it two
ways. You can take the Kociba data and predict the human response - what
would be elevated risks -- and it comes out right in the ballpark of
Fingerhut, Zober and Manz.  You can take the Fingerhut data and predict
what the animal tumour incidence would be, and it comes right out. In
fact, the Fingerhut data would actually predict more tumours in animals
than Kociba.

Tony Wagner, Environment Canada:  Your last slide in your off-the-record
comment, do some people take that as the first stop of organised
brinkmanship?

Linda Birnbaum: I'm not sure I know what organised brinkmanship means.

Tony Wagner: We are at a critical stage  either ban it or mount a
massive cleanup.

Linda Birnbaum: The last slide was the conclusion of our outside peer
panel. On the last day of the peer review, they actually sat there and
they listed effects, and they listed body burdens that were associated
with the effects in animals, and then they listed the body burdens
associated with effects in people. I should really stress, this is a
TEQ. If all you are worried about is dioxin, the levels of dioxin by
itself, are probably not that high. But when you look at the sum total
of what is out there, that is where the body burdens may be high enough
so you might say we are having a response. Your question about ban it or
not banning it I mean, what are you going to ban? We don't even know
where half this stuff is coming from. My feeling, and this is purely my
opinion, I have actually heard industry people say this, and this is
totally off the record, is that the total level of halogenated aromatic
organics in the environment is higher than they should be.

Denis Davis, Environment Canada:  Do you have the historic body burden
trends?

Linda Birnbaum: That is a really good question but we don't have the
information. Well, if you go back to Eskimo mummies from around 1600,
they were essentially non-detect. We don't have the information. There
is a suggestion, Larry Needham told me about a month ago that the CDC is
looking at the levels today.  Some of these levels are based upon
studies that were samples that were collected maybe 10 years ago, and
they think the levels are a little bit lower. But we don't have enough
numbers to really firm that up.

Doug Dodge, Ontario Ministry of Natural Resources: Is there any reason
to look at aboriginal people in a different way than we would look at
the rest of the general population, in terms of exposure and body
burden? Were they lumped into that group?

Linda Birnbaum: Yes they are. We know that the Inuit's in Hudson Bay
have much higher body burdens. Eric Dewailly from Quebec has actually
measured PCB levels and he has done it on a congener-specific basis and
they have about 10 times the TEQ.

Doug Dodge:  But could that similarly be a purpose for special
application in the Great Lakes, for example, where there are aboriginal
people just as dependent upon natural resources as the Inuits?

Linda Birnbaum: Yes -- you people are much more familiar -- I know there
is a Great Lakes Initiative where you are looking at levels, for
example, for certain Indian tribes and certain subsistence fisherman. In
New York State, they are looking at the Akwesasne tribe of Mohawk
Indians, and trying to determine levels, but I don't think their levels
are turning out to be higher than that of other people.  The reason that
the Inuits' levels are so high is because they eat sea mammals, and they
eat blubber, and I think there have been some comparisons done with the
Cree, who live in the same area, but they don't eat sea mammals. They
eat caribou and stuff like that, and their levels are not elevated. But
I think we are going to have questions of "what does it mean to be
elevated." Is two-fold enough? I don't know the answer to that. But, if
someone would say to me "what is a great big research need?" I would say
that we need to develop some sort of better way to do bio-monitoring,
because right now it costs $2,500 per sample to measure dioxins, furans
and PCB levels.  Well, you can't do a lot of samples and that is going
to rapidly deplete everyone's resources. We need to develop some sort of
methodology where we can get a measure which will be much more
sensitive, and much more cost effective, so that we can get a better
handle on "do we have populations where exposure is much higher than the
general population."

Doug Dodge: Things like the neurogenesis could be racially different,
could it not, so you look at different groups of people, you could get
different effect levels.

Linda Birnbaum: I think that is an absolute possibility. When you deal
with something like immunotoxicity, clearly it is a multi-genic factor.
Many things feed into how the immune system works -- it is very easy to
tickle it. I guess the Taiwanese data I find very important because they
do have people who are ethnically matched, but didn't eat the rice oil.
Much of the rice oil was eaten in a very limited province and at a
school in that province, so they really do have pretty good match
controls there, where that wouldn't really be much of a problem. I think
it is a problem with the Inuit study to find the appropriate controls.
The Cree, although they have a similar environment today in terms of
their housing and so on, are ethnically different.

Doug Dodge: I ask this question because some policy makers in the
Ontario Ministry of Natural Resources are under pressure from aboriginal
people to provide them with a selected "cleaner" source of native,
aboriginal-type foods and they are in special situations, because they
have different reactions and we have been "using the white man" as the
mode of subsistence.

Linda Birnbaum: There is no doubt, for example, that many aboriginal
peoples' diet is very different from the average white American or
Canadian diet. At our first public meeting we had a chief from some
tribe out west in Oregon, and basically said that the amount of fish
that his people ate were at least 10 times what the U.S. Food and Drug
Administration (U.S. FDA) estimates for fish consumption. So that
obviously is going to have a major impact on what the exposure is and,
very frankly, the exposure information is very limited. There has been
only one market basket, and it wasn't even a complete basket study done
in the entire U.S. There have been one or two done in Canada, so there
are some really big holes.
Thank you.

Compiled and Edited by Michael Gilbertson,
Former Secretary, Great Lakes Water Quality Board

Formatted by
Mary Ann Morin, Myrna Reid  and Bruce Jamieson

Many thanks to Bob McCray of Globe, Arizona, for sending Communities
Against Toxics the oridginal transcript enabling this important insight
to be shared among the communities of Britain and Europe. 


Please note there are a number of table and illustrations to go with the
text that I cannot post.



Regards

Ralph Ryder

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