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Effects of passive smoking and pregnancy

Institution: Lawrence Livermore National Laboratory
Investigator(s): Kenneth Turteltaub, Ph.D.
Award Cycle: 1999 (Cycle 8) Grant #: 8RT-0072 Award: $565,186
Subject Area: Cancer
Award Type: Research Project Awards

Initial Award Abstract
Studies in people have suggested that second-hand smoke from cigarettes can cause cancer in people who never smoked. Importantly, some of the most vulnerable non-smokers to second-hand cigarette smoke may be to newborn children. These studies, while implicating second-hand smoke as a human health threat, do not provide information on how this process occurs. Information on the way second-hand smoke causes cancer will aid significantly in our understanding of the risk this poses to the public and to children.

Smoking-related cancers are believed to involve chemicals present in the smoke. Passive smoke, which is emitted from the burning end of the cigarette, contains higher concentrations of these chemicals than main stream smoke. Upon inhalation, these chemicals pass from the smoke into the bloodstream and chemically bind to proteins and the genetic material of cells, DNA, forming adducts. DNA and protein adducts have been detected in the sperm of adult men who smoke and in the placenta and umbilical cords of newborn children of women who smoke. However, there has been no information to show that second-hand smoke causes this damage. There is also no direct method to measure these adducts at levels of chemical exposure equivalent to second-hand smoke.

The purpose of this work will be to specifically determine if DNA and protein adducts are formed when exposed to two chemicals present in cigarette smoke, nicotine and hydroquinone, at levels present in side-stream smoke. These chemicals are important components of the smoke. This will be possible using the novel and sensitive technique of accelerator mass spectrometry. This research will determine whether adduct levels increase with increasing dose and will help identify whether the adducts of these common components of cigarette smoke can be used to measure side-stream smoke exposure. This research will also help determine if this damage occurs in the offspring of smoking mothers.

Final Report
Passive smoking is a form of active smoking. Studies in people have suggested that second-hand smoke from cigarettes can cause cancer in people who never smoked. The chemicals released from the burning end of the cigarette have not been quantitatively assessed for health effects. Nicotine is a major chemical in cigarette smoke with more of it present in this “side-stream” smoke than in the inhaled main-stream smoke. The aim of this study has been to assess the tissue distribution and ability of nicotine to reach target tissues (bioavailability) and its ability to bind to DNA and protein at exposure levels equivalent to one cigarette. To measure bioavailability and adducts in specific target tissues, radioactive labeled nicotine (14C-labeled nicotine) was use as a tracer and the samples were analyzed by accelerator mass spectrometry (AMS), a novel and highly sensitive new technique.

To determine if nicotine reached specific tissues, mice were administered [14C]-nicotine by injection at a dose of 125 µg/ kg body weight. Tissues, protein and DNA were analyzed for [14C]-nicotine content between 0 - 48 h post exposure by measuring the 14C in tissues by liquid scintillation counting and in DNA and protein by accelerator mass spectrometry. The result showed that [14C]-nicotine was present in liver, lung, testis, brain and plasma with the highest levels found in the plasma, testis and liver followed by the lung and brain. Peak tissue levels were seen 15-60 minutes after exposure and declined thereafter. This established when nicotine was present in the tissues of interest. To assess the dose response for nicotine in the tissues, 14C-nicotine was administered to mice over a dose range spanning 75 µg/kg to 0.5 mg/kg body weight. [14C]-nicotine was measured at all these doses 1 hour following the exposure. The [14C]-nicotine could be quantified in tissue at levels as low as 20 ng of [14C]-nicotine per gram tissue. The greatest amount of nicotine was found in the liver followed by the kidney, lung, heart, and testis at all doses. Studies to assess whether DNA and protein binding was dose dependent revealed measurable adduct levels in protein from the liver, brain and testis at exposure levels of 75 or higher ug per kg body weight. Again, the liver had the highest level of binding, followed by the brain and testis. The nature of the dose responses for both the tissues and protein suggests a non-linear response. This work shows that nicotine is absorbed and distributed to various tissues at low doses. The data further shows that nicotine or its metabolites bind to protein and DNA at these low levels. Similar studies with hydroquinone (HQ) implied that it is also bioactivated and binds to liver and bone marrow protein and DNA. HQ treatment resulted in higher protein adducts in bone marrow compared to liver, which suggests that HQ concentrates in the bone marrow.

Further work to build on the present study will pursue the refinement of the dose responses curves in tissues, proteins and DNA. Detailed metabolite analysis should be undertaken to identify the bioavailable forms nicotine and elucidate mechanisms of its action on DNA and protein. Future expansion of the study will compare the effects of 14C-nicotine in female and male mice and in the developmental stages of mice for both protein and DNA damage in various tissues. Comparison of how nicotine is bioactivated at one cigarette equivalent exposure in pregnant mouse, new born and adult mice will provide needed data towards the understanding how dose influences bioactivation, and how the biomarkers used to measure chemical effects represent biological damage at low doses.