Abstract
This 1994 trip report by R.J. Reynolds chemist David J. Doolittle relates information on a multi-company operation called BESSE (Biological Effects of Sidestream Smoke Exposure), a project carried out conjointly by Philip Morris, R.J. Reynolds, British American Tobacco, the Verband (German cigarette industry), Austrian Tobacco and Swedish Tobacco. The goal of BESSE was "to strengthen our efforts to initiate and support science on biological effects of sidestream smoke exposure; to cooperate among the companies participating in the group; to switch from a reactive to a proactive approach in dealing with these [sidestream smoke] matters." (http://tobaccodocuments.org/pm/2023049185-9187.html)
The report says that one meeting participant (Dr. Franz Adlkofer) discusses a "wonderful, state-of-the-art" 90-day study on rats which revealed that secondhand smoke exposure caused "increased chromosome aberrations." The writer points out that this study, however, was never published in a peer-reviewed journal and was "unknown in the scientific world." According to Doolittle, Dr. Adlkofer then "told us that he wanted to do a 2-year study in rats, but the industry would not support the study because they were afraid of the results."
Doolittle talks about a proposed study "involving exposure of non-smokers to real life ETS in a bar for four hours." Doolittle says such a study would "not only be unhelpful, but would...leave the door open for prohibitionists to interpret it in a malicious manner...Therefore, I feel strongly that this study should not be done."
Doolittle mentions that it would be "ideal...to develop a data set which shows...the biological activity of NNK [a carcinogen in secondhand smoke] is inhibited by nicotine and cotinine...[and] the level of nitrosamines received by an individual following ETS exposure is insignificant compared to nitrosamine exposures from other sources, such as food."
Doolittle also relates a discussion about the "major problem" of international regulatory agencies considering ventilation standards that would require separate ventilation for smoking areas. He says:
"These draft regulations suggest that smoking not be banned, but areas that do have smoking would require separate ventilation. Thus, building owners would then have to decide whether or not they would provide separate ventilation for their smoking areas. Higher ventilation always results in more cost, both capital and operating. The problem is that owners of most establishments may decide to ban smoking, rather than pay a monetary penalty for enhanced ventilation equipment. Therefore, smoking would be a legal indoor activity, but no indoor locations could be found that meet the ventilation standards necessary to allow smokers to smoke. This is a very clever backdoor move by the prohibitionists, which must be aggressively countered with sound engineering, chemical and biological data."
Fields
- Quotes
3/1/94
Report on BESSE (Biological Effects of Sidestream Smoke Exposure) Meeting
The meeting on February 21 was attended by representatives from Swedish Tobacco (4 people), RJR (Doolittle, Green, Mueller), PM (4 people), BAT (2 people), Austrian Tobacco (1 person) and the VDC (3 people). The majority of these individuals remained silent throughout the day. I would like to share with you both objective and subjective impressions of the meeting.
Dr. Adlkofer (VDC) opened the meeting with (purportedly) a 2-hour review on ETS work done in the past. This review turned out to be a diatribe against the character and intelligence of people publishing in this area. No data were mentioned or discussed. These types of discussions are remarkably unproductive, and must be discouraged...He talked about the VDC 90-day rat study and what an outstanding study it was. He used 4 to 5 milligrams TPM [total particulate matter] per cubic meter, 10 hours a day, 5 days a week. He talked about the pathology they got,
dysplasia and metaplasia in rats, which was reversible. He claimed that he saw increased chromosome aberrations and increased SCEs in the rat lymphocytes. All in all, he described this as a wonderful, state-of-the-art study. However, it's curious that it has never been published in a peer reviewed journal and is unknown in the scientific
world. Dr. Adlkofer then told us that he wanted to do a 2-year study in rats, but the industry would not support the study because they were afraid of the results...He then went on to talk about the VDC bio-monitoring work and described how the VDC research has found no genotoxic effects in humans exposed to ETS, confirming {in his view) that ETS is not a health hazard. He had several examples where the VDC had obtained negative results in humans. I think many of us are well aware of the limitations of these studies, e.g. method sensitivity, which were not mentioned by Dr. Adlkofer. Again, it is relevant to note that none of
these studies (as far as I know) have ever been accepted for publication in peer-reviewed journals, and are virtually unknown in the scientific world...
...Unfortunately, the project aggressively championed by Dr. Adikofer was the documentation of exposure study (objective 1), involving exposure of non-smokers to
real life ETS in a bar for four hours. This is a reactive response to the American Health Foundation paper. The VDC would monitor nitrosamine levels in the air and verify that
people were, in fact, exposed to NNK. They would then collect their urine and measure NNAL and NNAL-glucuronide in the urine. I personally see little, if any, value
in this study and, perhaps unwisely, told Dr. Adlkofer so. In fact, I think this study would not only be unhelpful, but would probably be uninterpretable and leave the
door open for prohibitionists to interpret it in a malicious manner. Therefore, I feel strongly that this study should not be done...There are only two possible outcomes from the NNAL and NNAL-glucuronide
measurements - either you will fred it, or you won't find it. If you do find it, all it does is confirm the American Health Foundation thesis that non-smokers are exposed to
biologically relevant doses of NNK. We would not be able to put this into perspective in any way, nor be able to argue with any effectiveness that this level of exposure is
biologically insignificant, since we have no data to support this position. On the other hand, if we do not find the metabolites in the urine, there are only two possible
interpretations. The first interpretation is the NNK has been metabolized by the non-smoker to active metabolites and is now bound to the individuals' DNA, RNA and
protein. This obviously is an undesirable interpretation of the data. The other interpretation would be that the method is simply too insensitive to detect the metabolites at a low dose level. So in summary, there seems to be little, if any, value to these studies and Dr. Adlkofer was urged not to pursue these...
...In summary, in my mind the ideal would be to develop a data set which shows the following: First, people are exposed to very, very low levels of NNK in ETS. These
data will likely be developed by other individuals, so the industry needs (at most) to only confirm these numbers with limited studies. Second, the small amount of NNK
absorbed by individuals exposed to ETS is biologically insignificant for the following reasons: 1) It is effectively detoxified and excreted by the body. 2) The biological activity of NNK is inhibited by nicotine and cotinine which will always be present in gross excess relative to NNK, both in atmospheres and in the body. 3) The level of nitrosamines received by an individual following ETS exposure is insignificant compared to nitrosamine exposures from other sources, such as food.
Next, we discussed the international ventilation standards. This could be a major problem because such ventilation standards are apparently being considered by regulatory agencies around the world. These draft regulations suggest that smoking not be banned, but areas that do have smoking would require separate ventilation. Thus, building owners would then have to decide whether or not they would provide
separate ventilation for their smoking areas. Higher ventilation always results in more cost, both capital and operating. The problem is that owners of most establishments may decide to ban smoking, rather than pay a monetary penalty for enhanced ventilation equipment. Therefore, smoking would be a legal indoor activity, but no
indoor locations could be found that meet the ventilation standards necessary to allow smokers to smoke. This is a very clever backdoor move by the prohibitionists, which
must be aggressively countered with sound engineering, chemical and biological data.
- Company
- R.J. Reynolds
- Author
- *Bethizy, J. Donald (use DeBethizy, Joseph Donald, Ph.D.) (Head of product evaluation for RJR, c. 1994)
Defense
- Recipient
- Blixt, Charles A., - Assistant General Counsel for R. J. Reynolds Tobacco, c. 1994
- Burger, Gary Thomas - Vice President of Product Development for R. J. Reynolds Tobacco Company in 1994.
- Caldwell, Bill-involved with Sidestream and ETS Nitrosamine Analyses for RJR
- Coggins, Christopher-Toxicologist for RJR
- Bethizy, J. Donald -Head of Product Evaluation for RJR Circa 1994.
- Green, Charles R. PhD - RJR Chemical Division 1969, Principal Scientist 1989, Senior Principal Scientist R&D 1997, worked on BESSE project (Biological Effects of Sidestream Smoke Exposure).
- Lippiello, Patrick - Scientist, RJR
- Mosberg, Amie
- Rees, Cooper
- Robinson, John - Research scientist and psychologist for RJR, c. 1994
- Suber, Robert - Spokesperson for RJR
- Ward, Mary - Associate General Counsel for RJR
- Region
- United States
- Sweden
- Austria
- Germany
- Named Organization
- American Health Foundation (Health Research)
Plaintiff
- Named Person
- Swedish Tobacco
- RJR, R.J. Reynolds Tobacco Company
- Doolittle, D.J.
- Philip Morris
- B&W, Brown & Williamson
- Austrian Tobacco
- Verband
- Adlkofer, Franz (VDC Research Director)
Argued in a 1988 joint meeting of the worldwide tobacco companies that the industry should stop developing "marketable science" for use in public relations to fight the secondhand smoke issue and instead should establish a safe threshold for exposure to secondhand smoke. (Bates No. 2021548222/8235)
- Mueller, L.
- *Green, Charles R. (use Green, Charles Raymond, Ph.D.) (RJR Chemical Division, c. 1969)
- Carchman, Richard Allan, Ph.D. (PM Scientific Affairs, Research & Development VP)
Philip Morris Director and worked in Scientific Affairs. Reported to Cathy ellis.
- American Health Foundation
- Lee, C.
- Scherer
- Tricker
- Poulsen, H.
- Risner, C.
- Rajewsky
- University of Copenhagen
- Hoffman Laroche
- BAT, British American Tobacco
- Krone, D.
- Finnish Academy of Science
- Ferrosan
- Loft, S.
- Ames, B.
- Skakkebaek, N.E.
- WHO, World Health Organization
- NIH, National Institutes of Health
- Wyrobek, A.
- Operation/Project
- BESSE - Biological Effects of Sidestream Smoke Exposure
- Type
- REPORT
- Subject
- secondhand smoke
- biologically active substance
- research activity
- industry activity
- industry recommendation
- industry response
- industry sponsored research
- industry strategy
- Ventilation (Design)
Document Images
Page 1: qix83d00
Date: March 8, 1994
To: DISTRIBUTION
From: David J. Doolittle
Subj: Trip Reports
is
I have attached several trip reports I thought may interest you.
DJD:kfyAriprptrnem
DISTRIBUTION:
Chuck Blixt
Gary Burger
Bill Caldwell
Chris Coggins
Don deBethizy
Charlie Green
Brad Ingebrethsen
Pat Lippiello
Arnie Mosberg
Cooper Rees
John Robinson
Bob Suber
Mary Ward
CMB Division Members
Page 2: qix83d00
3/1/94 - _
Reuort.on BESSE (Biological Effects of Sidestream Smoke Exposure) Meeting
The meeting on February 21 was attended by representatives from Swedish Tobacco
(4 people), RJR (Doolittle, Green, Mueller), PM (4 people), BAT (2 people), Austrian
Tobacco (1 person) and the VDC (3 people). The majority of these individuals
remained silent throughout the day. I would like to share with you both objective and
subjective impressions of the meeting.
Dr. Adikofer (VDC) opened the meeting with (purportedly) a 2-hour review on ETS
work done in the past. This review turned out to be a diatribe against the character
and intelligence of people publishing in this area. No data were mentioned or
discussed. These types of discussions are remarkably unproductive, and must be
discouraged.
Dr. Adikofer then moved on to a discussion of the VDC research that he had done over
the last ten years. He first talked about a planned prospective study that he proposed
on ETS that was going to look at misclassification and so forth; however, he said that
he was never funded by the industry to do this. He did tell us that the WHO MONICA
study, which was a misclassification study, did show that 17% of ex-smokers claim
they are life-long non-smokers. To me this was a high number which should be
pursued. He then talked about the VDC animal studies. He talked about the VDC 90-
day rat study and what an outstanding study it was. He used 4 to 5 milligrams TPM per
cubic meter, 10 hours a day, 5 days a week. He talked about the pathology they got,
dysplasia and metaplasia in rats, which was reversible. He claimed that he saw
increased chromosome aberrations and increased SCEs in the rat lymphocytes. All in
all, he described this as a wonderful, state-of-the-art study. However, it's curious that it
has never been published in a peer reviewed journal and is unknown in the scientific
world. Dr. Adlkofer then told us that he wanted to do a 2 year study in rats, but the
industry would not support the study because they were afraid of the results. Dr.
Adlkofer then talked about the VDC analytical work. He said he has done some, but
didn't discuss the data. He then went on to talk about the VDC bio-monitoring work
and described how the VDC research has found no genotoxic effects in humans
exposed to ETS, cont'irming (in his view) that ETS is not a health hazard. He had
several examples where the VDC had obtained negative results in humans. I think
many of us are well aware of the limitations of these studies, e.g. method sensitivity,
which were not mentioned by Dr. Adlkofer. Again, it is relevant to note that none of
these studies (as far as I know) have ever been accepted for publication in peer-
reviewed journals, and are virtually unknown in the scientific world.
Page 3: qix83d00
We then toofC a coffee break. During the break, I discussed with Charlie and Lutz the
possibility that perhaps I could give a short lecture on basic toxicological principles to
help focus the discussion. Charlie and Lutz agreed that this would be appropriate, so
after the break, I gave an impromptu lecture on threshold and non-threshold dose
response curves, the difference between no-observed-effect-levels and biological
thresholds, and other rudimentary aspects of toxicological risk assessment
procedures. This elementary lecture was followed by a surprising amount of
discussion. I believe that the question of differentiating a true threshold from a no-
observed-effect-level due to limitations in method sensitivity had never been
considered by the VDC. It is an interest to note that during this discussion Dr.
Carchman, the leader of the Philip Morris contingent, expressed extremely strong
support for the need to stop the delay tactics and to stop criticizing other peoples'
studies and morality. Dr. Carchman clearly indicated that the industry needs to do
studies of high scientific quality and to publish these studies in peer reviewed journals!
He emphasized the need to get out of our defensive posture and enter into a more
productive dialog.
After the lunch break, Dr. Lutz Mueller suggested that we hold discussion on the VDC
Human ETS Study until we had finished the discussion on nitrosamines, since the
nitrosamine questions in his view would be easily resolved and we could move on and
discuss the Human ETS studies. Unfortunately, the nitrosamine studies turned out to
not be easily resolved and we spent several hours discussing them. The VDC has
proposed several studies. I will be happy to provide the original. document to anyone
who would like it. Briefly, there are four objectives. The first objective is
DEfERMINATIOiv OF REAL LIFE EXPOSURE TO NNK. The second objective is EVALUATION OF ETS
EXPOSURETO NNK UNDER CONTROLLED EXPERIMENTAL CONDITIONS. The third objective is
ASSESSMENT OF URINARY NITROSAMINE EXCRETIONS FROM OTHER EXPOSURE SOURCES and the
fourth objective is ANIMAL STUDIES AND GENETIC TOXICOLOGY STUDIES. The RJR position is
that the first two objectives, documenting NNK exposure, are not helpful in the
absence of biological data. Therefore, our suggestion was that the studies on
understanding the biological effect(s), if any, of NNK exposure be moved up in
importance.
Unfortunately, the project aggressively championed by Dr. Adlkofer was the
documentation of exposure study (objective 1), involving exposure of non-smokers to
real life ETS in a bar for four hours. This is a reactive response to the American Health
Foundation paper. The VDC would monitor nitrosamine levels in the air and verify that
people were, in fact, exposed to NNK. They would then collect their urine and
measure NNAL and NNAL-glucuronide in the urine. I personally see little, if any, value
in this study and, perhapi unwisely, told Dr. Adlkofer so. In fact, I think this study
would not only be unhelpful, but would probably be uninterpretable and leave the
door open for prohibitionists to interpret it in a malicious manner. Therefore, I feel
strongly that this study should not be done.
Page 4: qix83d00
There are`only two possible outcomes from the NNAL and NNAL-glucuronide
measurements - either you will find it, or you won't find it. If you do find it, all it does
is confirrm'the American Health Foundation thesis that non-smokers are exposed to
biologically relevant doses of NNK. We would not be able to put this into perspective
in any way, nor be able to argue with any effectiveness that this level of exposure is
biologically insignificant, since we have no data to support this position. On the other
hand, if we do not find the metabolites in the urine; there are only two possible
interpretations. The first interpretation is the NNK has been metabolized by the non-
smoker to active metabolites and is now bound to the individuals' DNA, RNA and
protein. This obviously is an undesirable interpretation of the data. The other
interpretation would be that the method is simply too insensitive to detect the
metabolites at a low dose level. So in summary, there seems to be little, if any, value
to these studies and Dr. Adlkofer was urged not to pursue these (however, I personally
believe that he will).
My strong recommendation was that the VDC focus on the 4th objective, assessment
of biological activity. They were encouraged to synthesize NNAL and NNAL-
glucuronide and perform some biological testing, the idea being to confirm that these
are non-toxic metabolites of NNK. The industry could then thank the American Health
Foundation for showing that individuals exposed to ETS effectively detoxify NNK and
rapidly excrete the detoxified metabolites, resulting in no biological activity. I also
recommend that the VDC studies on putting the exposure of NNK in perspective with
other nitrosamines (Objective 3) be conducted. Finally, I recommended that the
studies by Dr. Chin Lee showing that nicotine and cotinine inhibit the biological activity
of NNK be aggressively expanded.
In summary, in my mind the ideal would be to develop a data set which shows the
following: First, people are exposed to very, very low levels of NNK in ETS. These
data will likely be developed by other individuals, so the industry needs (at most) to
only confirm these numbers with limited studies. Second, the small amount of NNK
absorbed by individuals exposed to ETS is biologically insignificant for the following
reasons: 1) It is effectively detoxified and excreted by the body. 2) The biological
activity of NNK is inhibited by nicotine and cotinine which will always be present in
gross excess relative to NNK, both in atmospheres and in the body. 3) The level of
nitrosamines received by an individual following ETS exposure is insignificant
compared to nitrosamine exposures from other sources, such as food.
Next, we discussed the international ventilation standards. This could be a major
problem because such ventilation standards are apparently being considered by
regulatory agencies around the world. These draft regulations suggest that smoking
~,
not be banned, but areas that do have smoking would require separate ventilation.
Thus, building owners would then have to decide whether or not they would provide o
separate ventilation for their smoking areas. Higher ventilation always results in more ~
cost, both capital and operating. The problem is that owners of most establishments ~ i
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Page 5: qix83d00
may decide xo ban smoking, rather than pay a monetary penalty for enhanced
ventilation equipment. Therefore, smoking would be a legal indoor activity, but no
indoor locations could be found that meet the ventilation standards necessary to allow
smokers to smoke. This is a very clever backdoor move by the prohibitionists, which
must be aggressively countered with sound engineering, chemical and biological data.
It was now approximately 5:00 p.m. and the VDC scientists, Dr. Adlkofer, Dr. Scherer
and Dr. Tricker had to catch an airplane back to Germany. Therefore, most
unfortunately, we had no time to discuss the VDC Human ETS study. Dr. Scherer did
distribute a rough protocol for the study, which I have. The plan is that we will
reconvene at some later time (May was proposed) to discuss this proposal which is
entitled "Expo'94". I would be happy to provide this protocol to anyone who is
interested.
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Additional Comments and Observations:
1. I discussed with Dr. Scherer his progress on the 8-OHdGuanosine assay. He is at
exactly the same point that RJR is, in that he has a single column method that is
very appropriate for analyzing the adducts when they are in DNA. He too has
struggled with the techniques for cleaning up the urine sufficiently to analyze the
adduct in urine. He has tried the triple-column method of Poulsen and been
unsuccessful in getting it to work. This is consistent with Charlie Risner's
comments to us that a triple-column is very difficult to do. Dr. Scherer also told me
that a colleague of his actually visited Dr. Poulsen's laboratory and spent several
weeks there, came back to the laboratory and discovered that he could not get the
assay going in his own laboratory because of the complexity. As indicated in the
Poulsen trip report (attached), Dr. Poulsen's technician is a middle-aged woman
with many years of experience. She is truly a HPLC expert and has uniquely
mastered this particular technique. My current view is that affinity chromatography
may be the best option for cleaning up the urine. Dr. Scherer is proceeding on this
by giving Dr. Rajewsky a contract to develop the 8-OHdG antibodies which he is
apparently doing, and of course, I will be pursuing it with Dr. Ames, who has
already offered to make his antibodies available to us. I will continue to stay in
contact with Dr. Scherer as we continue to work together to solve this important
problem.
2. I also discussed the VDC human urine samples from the. ETS. study conducted in
Germany. This is a study where the VDC went to smokers' and non-smokers'
homes and very carefully measured the air quality in these homes and also very
carefully measured the nicotine exposure of both smokers and non-smokers
exposed to ETS and non-smokers who were not exposed to ETS. The VDC has in
their possession very limited volumes of urine from these individuals. Dr. Scherer
agreed, after listening to our experimental design, to make those available to us to
assay for urine mutagenicity. Our hypothesis is that under very tightly controlled
laboratory conditions, one can see a very, very slight increase in urinary
mutagenicity following exposure to high levels of ETS. However, in the real world,
where people eat varied diets and are exposed to various other materials in the air
and where the ETS exposure would be much lower, ETS has no discernible effect
on urinary mutagenicity. I also described to Dr. Scherer our ideas on thromboxane
and prostacyclin metabolites and told him that we would like to measure these in
the urine samples. He was agreeable to us performing those studies. He will be
shipping the samples to us within the next 2 weeks. Our laboratory will work to get
them done in a timely manner and the results should be available in the near
future. One word of caution - the amount of urine available to us, which is
somewhere between 65 and 70 ml per individual is a very limited amount for these
types of analyses. Therefore, we are going to have one chance to do the assay at
one dose lev4
Page 7: qix83d00
3/1/94 _ David J. Doolittle
Trip Report of Meeting with Dr. Henrik Poulsen at the University of Copenhagen.
Dr. Poulsen is an internationally recognized expert in the area of oxidative stress, with
particular reference to its effects on human health. Dr. Poulsen has both a M.D. and a
Ph.D. degree and is uniquely qualified to discuss both the basic science and the
clinical pathology of this important problem.
Please recognize that we and Dr. Poulsen have signed a confidentiality agreement and
therefore he was able to share confidential information with me. The information
which I will be passing on to you should be regarded as SECRET. and must not be
shared, esaecially with Hoffman-LaRoche.
The first study that Dr. Poulsen and I discussed was a study which has been funded, in
part, by the British American Tobacco Company (BAT). They have given Dr. Poulsen a
grant of $2,000,000 Danish Krone (approximately $300,000). This study is being done
in collaboration with several groups, including a group with the Finnish Academy of
Science and a vitamin company in Scandinavia called Ferrosan. Ferrosan is supplying
all the mitigants for this study and would like to use the results of this study in their
advertising. This study is an intervention study in 140 heavy smokers. Each one of
these individuals is smoking more than 20 cigarettes per day. There are 3 mitigants
being used: Vitamin E (100 I.U. twice a day), Vitamin C (250 mg. twice a day), and Q10
(30 mg. three times a day). The mitigants will be taken for 8 weeks. These studies are
being done using each mitigant separately, and also the various mitigants in
combination, so there are a total of 7 groups of heavy smokers with 20 people in each
group. There are no non-smokers in this study. Baselines have been taken and the
study started on February 21. It will be a staggered start, so they will not complete the
study for 4 months, which means it will be completed on June 21 st. There are no
dietary controls; however, the subjects are being instructed not to take dietary
supplements and just to eat their normal diet. They are being given a questionnaire to
fill out on their diet.
The objective of the study is to determine whether oxidative stress in smokers can be
mitigated by administration of these vitamins. There are several endpoints: 1)
Measurement of 8-OHdGuanosine in the urine. 2) Measurement of plasma levels of
Vitamin E, C and Q 10. 3) Examination of LDL. The LDL examination will indude
endpoints such as the concentration of Vitamin E in LDL, concentration of Q10 in the
LDL, as well as the lag time and the amount of peroxy radicals in the LDL
Page 8: qix83d00
I would like -to tell you a little bit more about the LDL analysis, since it is a new
technique that I have not had a chance to tell many of you about. LDL contains an
ApoB protein. This ApoB protein is a cellular receptor on the LDL molecule. When
this ApoB protein is oxidized, scavenger macrophages bind to the oxidized ApoB
receptor protein on the LDL and take up the LDL. These scavenger macrophages,
which are then termed foam cells due to foaming appearance of the fat in their
cytoplasm, migrate into the arterial wall to form fatty streaks. These fatty streaks then
cause some toxicity in the cell wall which results, according to the current theory, in
an artherosclerotic plaque. The current theory is that free radicals oxidize the lipids in
LDL and form peroxides, which then proceed as a chain reaction within the
cholesterol in the LDL molecule, finally resulting in oxidation of the ApoB receptor
protein. So, in order to measure the level of oxidation in the LDL, one uses a reaction
called a "lag time assay". The lag time assay is based on the amount of time that it
takes for LDL to oxidize in the test tube after you add either copper (or iron), which, of
course, will initiate a Fenton reaction. The more unoxidized Vitamin E in the LDL, the
longer the lag time; in other words the longer that the LDL is protected from oxidation
via the Fenton reaction the longer the lag time. There are approximately 10 Vitamin E
molecules in an LDL particle under normal circumstances. This may be a technique
which we may want to add at some point, since it directly addresses the heart disease
issue.
There is a second part to the BAT study which will not start until September 1, after the
first study is complete. This is a smoking cessation study. The endpoints to be
measured are the same as in the first study, except in this study, there is no
supplementation. The subjects will have their oxidative stress levels measured before
they stop smoking, and then at various times after they stop smoking. The hypothesis
is that the oxidative stress will come down when people stop smoking. There is an
interesting twist to this study. As many of you know, Dr. Poulsen believes that a
substantial amount of the oxidative stress in smokers is caused by nicotine raising the
basal metabolic rate. Therefore, some of these smokers in the smoking cessation
study will be given nicotine patches. He is going to compare the level of oxidative
stress in smokers, ex-smokers with nicotine patches, and ex-smokers without nicotine
patches. His hypothesis is that smokers that have the nicotine patch will have nearly
the same level of oxidative stress as the smokers, if there can be enough nicotine
absorbed (this is a BIG if).
Dr. Poulsen's plan, after he completes the initial portion of the BAT study which
involves supplementing the smokers with these relatively high doses of supplements,
is to perform a dose response curve using the most effective supplement(s). This is
very important for the vitamin company, as well as probably for us, since it will tell us
how much mitigant the smoker has to receive in order to reduce their level of
oxidative stress. -
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Page 9: qix83d00
Dr. Poulse`n and I then discussed a very confidential study, funded by Hoffman-
LaRoche, on the effect of beta carotene intervention on oxidative stress in smokers
(more than 10 cigarettes a day). The smokers were given either a placebo or 20
milligrams of beta carotene a day for 8 weeks. Neither the subject nor the physician
knew which was placebo and which was beta carotene. There were 57 smokers who
received beta carotene and 65 who received placebo. The most surprising result of
this study, (although perhaps not too surprising for many of us who recognize the
mechanism of action of beta carotene), was that there was no difference in the
amount of 8-OHdGuanosine excreted in the urine between the placebo or beta
carotene groups. This data clearly suggests that beta carotene alone does not reduce
oxidative stress in smokers. I personally reviewed the raw data with Dr. Poulsen and I
can tell you the data are pretty tight. They do have such things as beta carotene
concentrations in the plasma, which were substantial, so the people did take their
piIls.
The other piece of information which came out of this study which was perhaps a little
surprising to me, was that there was no relationship between the amount of
8-OHdGuanosine in the urine and the subjects' cigarettes per day (10-25) or the
subjects' number of pack years. These data were true with both the placebo and the
beta carotene-supplemented smokers. This has some implications for our mitigant
project. These results appear to support Poulsen's hypothesis that nicotine is a major
factor in inducing oxidative stress by increasing basal metabolic rate. His view, and I
don't know how true this is, is that smokers reach an elevated BME rather rapidly and
increasing the number of cigarettes smoked in a day does not substantially increase
this basal metabolic rate. Of course, a flat dose-response curve could also be the
result of chronic inflammation (which would produce oxidative stress). Ten cigarettes
per day may induce a level of chronic inflammation similar to 20 cigarettes a day.
Determining the relative importance of smoke chemistry, chronic inflammation and
nicotine in oxidative stress would appear to be of great importance to RJR, since this
would dramatically affect product development approaches to mitigation.
Finally, Dr. Poulsen told me of a study which he has done in non-smokers where he
measured urinary 8-OHdGuanosine after vegetable ingestion. These results, in my
view, are most unfortunate in that the only effective mitigant for reducing urinary
.8-OHdGuanosine in non-smokers were the dreaded brussel sprouts. Daily brussel
sprouts decreased urinary 8-OHdGuanosine by 28%. There was no effect with
broccoli, and there was no effect with non-cruciferous vegetables. So forget about
eating your broccoli and salads - eat your brussel sprouts.
More bad news for gourmets-- Poulsen has unpublished data in rats clearly showing
that fat (both saturated and unsaturated) substantially increases oxidative stress.
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I discussed-the triple column method with Dr. Poulsen, examined the equipment in his
laboratory, and watched his technician performing the assay. My conversations with
Dr. Poulsen's technician, who appears to be extraordinarily experienced and
competent, indicates that on a good day if everything goes correct, the sample
through-put is one urine sample per day. There are many things that can, and often do
go wrong. Therefore, it appears as if this method is inappropriate for use at RJR. The
sample through-put is simply too slow for our purposes. I met with Dr. Stephen Loft,
an HPLC expert who works with Dr. Poulsen. Loft is currently trying other columns
and approaches in order to speed up the method. He promised to call me if he is
successful.
During a tour of Poulsen's laboratories, I met a postdoctoral fellow who has
transfected human P450s into V79 cells. I described Chin Lee's work, showing that
nicotine and cotinine inhibit the mutagenicity of NNK, presumably via competitive
inhibition of cytochrome P450. Dr. Poulsen told me that we could use his cell lines to
determine which P450 enzyme was involved.
