Date:
September 15,
2014
Source:
Oregon
State University
Sunflower seeds and oil are a particularly good dietary source of
vitamin E.--Amid
conflicting reports about the need for vitamin E and how much is enough,
a new analysis published today suggests that
adequate levels of this essential micronutrient are especially critical
for the very young, the elderly, and women who are or may become
pregnant.-A lifelong proper intake of vitamin E is also important,
researchers said, but often complicated by the fact that this nutrient
is one of the most difficult to obtain through diet alone. It has been
estimated that only a tiny fraction of Americans consume enough dietary
vitamin E to meet the estimated average requirement.-Meanwhile,
some critics have raised unnecessary alarms about excessive vitamin E
intake while in fact the diet of most people is insufficient,
said Maret Traber, a professor in the College of Public Health and Human
Sciences at Oregon State University, principal investigator with the
Linus Pauling Institute and national expert on vitamin E.-"Many people
believe that vitamin E deficiency never happens," Traber said. "That
isn't true. It happens with an alarming frequency both in the United
States and around the world. But some of the results of inadequate
intake are less obvious, such as its impact
on things like nervous system and brain development, or general
resistance to infection."-Some of the best dietary sources of
vitamin E -- nuts, seeds, spinach, wheat
germ and sunflower oil -- don't generally make the highlight
list of an average American diet. One study found that people who are
highly motivated to eat a proper diet consume almost enough vitamin E,
but broader surveys show that 90 percent of men and 96 percent of women
don't consume the amount currently recommended, 15 milligrams per day
for adults.--In a review of multiple studies, published in Advances
in Nutrition, Traber outlined some of the recent findings about
vitamin E. Among the most important are the
significance of vitamin E during fetal development and in the first
years of life; the correlation between adequate intake and dementia
later in life; and the difficulty of evaluating vitamin E
adequacy through measurement of blood levels alone.
Findings include:
- Inadequate vitamin E is associated with increased infection,
anemia, stunting of growth and poor outcomes during pregnancy for both
the infant and mother.
- Overt deficiency, especially in children, can cause neurological
disorders, muscle deterioration, and even cardiomyopathy.
- Studies with experimental animals indicate that vitamin E is
critically important to the early development of the nervous system in
embryos, in part because it protects the function of omega-3 fatty
acids, especially DHA, which is important for brain health. The most
sensitive organs include the head, eye and brain.
-
One study showed that higher vitamin E concentrations
at birth were associated with improved cognitive function in
two-year-old children.
- Findings about diseases that are increasing in the developed
world, such as non-alcoholic fatty liver disease and diabetes, suggest
that obesity does not necessarily reflect adequate micronutrient
intake.
- Measures of circulating vitamin E levels in the blood often rise
with age as lipid levels also increase, but do not prove an adequate
delivery of vitamin E to tissues and organs.
- Vitamin E supplements do not seem to prevent Alzheimer's disease
occurrence, but have shown benefit in slowing its progression.
-
A report in elderly humans showed that a lifelong
dietary pattern that resulted in higher levels of vitamins B,C, D and
E were associated with a larger brain size and higher cognitive
function.
-
Vitamin E protects critical fatty
acids such as DHA throughout life, and one study showed that people in
the top quartile of DHA concentrations had a 47 percent reduction in
the risk of developing all-cause dementia.
"It's important all of your life,
but the most compelling evidence about vitamin E is about a 1000-day
window that begins at conception," Traber said. "Vitamin E is critical
to neurologic and brain development that can only happen during that
period. It's not something you can make up for later."--Traber said she
recommends a supplement for all people with at least the estimated
average requirement of vitamin E, but that it's particularly important
for all children through about age two; for women who are pregnant,
nursing or may become pregnant; and for the elderly.-Story
Source--The above story is based on
materials provided by
Oregon State University. Note: Materials may be
edited for content and length.---Journal Reference--Maret
Traber. Vitamin E Inadequacy in Humans: Causes and Consequences.
Advances in Nutrition, September 2014
******************************************************************
-
Maret G. Traber*
1.
School of
Biological & Population Health
Sciences
and the Linus Pauling
Institute,
Oregon
State
University,
Corvallis, OR
-
Danny Manor
+ Author Affiliations
1.
Departments
of Nutrition and of Pharmacology, School of Medicine,
Case
Western
Reserve
University,
Cleveland,
OH
Vitamin E refers
to the plant-derived,
lipid-soluble antioxidants:
tocopherols and tocotrienols.
They terminate
the chain reaction
of lipid peroxidation.
Vitamin
E biological activity is different
from its antioxidant activity, and there
is a preference
for α-tocopherol. This preference
is achieved
through the selective
degradation and
excretion
of other
vitamin
E forms and the
selective
retention
of α-tocopherol, mediated
by the hepatic
α-tocopherol transfer
protein (α-TTP). Hepatic
α-TTP facilitates the
selective
incorporation of α-tocopherol
into circulating lipoproteins
that distribute the
vitamin to nonhepatic
tissues. α-TTP is therefore
considered
to be the
major regulator of
vitamin
E status in humans.
Next Section
Deficiencies:
Vitamin
E deficiency
occurs in humans as a result
of genetic
abnormalities in α-TTP or in
lipoprotein synthesis
or occurs secondary to fat
malabsorption. Genetic
defects
in α-TTP are associated
with a characteristic syndrome,
ataxia with vitamin
E deficiency
(AVED). More
than 20 mutations in α-TTP have
been identified
in human AVED patients.
Diet
recommendations:
The
vitamin
E dietary
reference
intake (DRI) for adult men
and women (and individuals
14–18 y) was set
in 2000 at a daily estimated
average
requirement
(EAR) of 12 mg α-tocopherol
and an recommended
daily allowance (RDA) of 15
mg.
There
are no increases
for pregnancy, but for
lactation the RDA is 19 mg/d.
The adequate
intake (AI) for infants (0–6
mo) was estimated
to be 4 mg and for 7 through
12 mo to be 5 mg. The
RDA for children 1 to 3 y is
6 mg; for those 4–8 y, it is
7 mg and 11 mg for those 9 to
13 y. --Plant-synthesized
α-tocopherol is RRR-α-tocopherol;
chiral carbons are in the
R-conformation at positions 2, 4′, and 8′. Chemical
synthesis results
in an equal mixture
of 8 different
stereoisomers
(RRR, RSR, RRS, RSS, SRR,
SSR, SRS, SSS) that is called
all-rac-α-tocopherol.
The 2 position of α-tocopherol,
the junction of the
ring and tail, is critical for in vivo α-tocopherol
vitamin activity. Only 2R
forms meet human requirements.
Food sources:
Major dietary
vitamin
E sources,
as commonly eaten
portions [USDA National Nutrient
Database for Standard Reference,
Release
24 (January 2012;
http://www.nal.usda.gov/fnic/foodcomp/search/)]
are fortified
ready-to-eat
cereals;
nuts, especially
almonds; seeds, such as
sunflower seeds;
greens, such as spinach; and
vegetable
oils, especially
sunflower and safflower.
Clinical uses:
Humans with defects
in the
TTPA
gene
(encoding α-TTP) have
extraordinarily low (1/100 of
normal) plasma vitamin
E concentrations,
but if they
are given
vitamin
E supplements,
plasma concentrations
normalize within hours; if supplementation
is halted, plasma
vitamin
E concentrations
decrease
within days to deficient
levels.
A daily α-tocopherol dose
(800–1200 IU) is usually sufficient
to prevent
further deterioration
of neurologic function, and
in some cases,
improvements
have been
noted. Postmortem
analysis of a brain from a vitamin
E–supplemented
AVED patient
demonstrated
vitamin
E accumulation and prevention
of Purkinje cell
loss. -Vitamin
E deficiency
due to impaired
lipoprotein synthesis
or fat malabsorption syndromes
(e.g., abetalipoprotenemia,
cystic fibrosis, short bowl disorder,
choleastasis, and inherited
defects
in bile acid synthesis)
is also treated
with daily vitamin
E supplements
(100 mg/kg body weight). --The
benefit
of vitamin
E supplements
in individuals who are not
vitamin
E deficient
is controversial. In the
elderly,
impaired immune
function was improved with
vitamin
E supplementation.
Patients with macular degeneration
benefited
from a supplement
cocktail that included
vitamin
E.
Vitamin
E supplements
have decreased
heart attack risk in those
with the haptoglobin 2–2 genotype,
which results in a
dysfunctional protein and
causes increased
oxidation by free heme.
A follow-up study of the
Alpha-Tocopherol Beta-Carotene
Cancer Prevention
trial showed that men
at baseline
who consumed 12 mg α-tocopherol
daily (equivalent
to the
vitamin
E
EAR)
had considerably lower
risk of total and cause-specific
mortality 13 y later.
Toxicity:
The
upper limit of tolerable
intakes (UL) is 1000 mg/day,
equivalent
to 1100 iu synthetic
or 1500 iu natural
vitamin
E. The
UL was based on the
adverse
effect
of an increased
bleeding tendency
observed
in rat studies. This tendency
to bleed was found to have
beneficial
effects
in preventing
venous thrombosis in a trial
of vitamin
E supplements
in women. --Subsequent
to the publication of the
DRI, there
have been
several
meta-analyses
evaluating the
outcomes of human
vitamin
E supplementation
trials. Thus far, there
have been
no uniform mechanisms identified
for the claim in meta-analyses
of increased
mortality associated with
vitamin
E supplements.
Previous SectionNext
Section
Recent
research
Vitamin
E in the
central nervous
system:
Vitamin E deficiency
manifests primarily as cerebellar
ataxia in humans, underscoring
the unique
sensitivity of the
central nervous
system to oxidative
stress.
Interestingly,
α-TTP is expressed
in the cerebellum.
α-Tocopherol supplementation
in α-TTP knockout mice
normalizes its status in all
tissues
except
the brain. Thus, a unique
relationship
exists between
localized
vitamin
E levels,
expression
of α-TTP, oxidative stress,
and optimal cerebellar
function. The detailed
description of this relationship
and the molecular
mechanisms that underlie
it are critical research
questions.
Homeostasis:
An important fundamental
question in
vitamin
E biology is “are
there
compensatory mechanisms
in response
to oxidative stress
to increase
α-tocopherol concentrations
or distribution?.” Provocatively,
brain α-TTP expression
levels
respond to oxidative
stress,
but liver
α-TTP does not.
Fertility:
Vitamin
E was discovered
as an essential
dietary factor for reproductive
health in female
rodents. Surprisingly, very
little is known regarding
human vitamin
E status and reproductive
health.
In mice,
α-tocopherol sufficiency
is essential
for placentation. α-TTP is
expressed
in the uterine
wall of pregnant female
mice and in the
human placenta. Research
in this field is of great
importance because
96% of U.S. women do not meet
vitamin
E
EAR.
Interactions
with other nutrients:
The
untoward effects
of vitamin
E supplements
on blood clotting may result
from vitamin
E and K interactions
because
these
supplements
increase
undercarboxylation of
prothrombin, suggesting lower
vitamin K activity. More
than 90% of dietary, plant-derived
vitamin K is phylloquinone
(vitamin K1) with a 20-carbon
phytyl side chain that is identical
to that of tocopherol.
Phylloquinone is converted
to menadione
and then to MK-4 (extrahepatic
tissue
vitamin K). Based
on their similar structures,
vitamins
E and K likely
share the
same pathways for metabolism
and excretion.
Possible mechanisms
for the
vitamin
E and K interaction
have been
proposed, but none
have been
proven.
The
interactions of
vitamins
E and C are
likely dependent
on their roles
as antioxidants; vitamin C
can regenerate
tocopherol from the
tocopheroxyl radical. When
α-tocopherol kinetics
were
evaluated
in cigarette
smokers, smokers
had higher F2-isoprostane
concentrations and faster
plasma α-tocopherol disappearance
rates than nonsmokers.
When smokers
received
vitamin C supplementation
(500 mg twice daily) for 2 weeks,
α-tocopherol disappearance
rates were
normalized. Thus, in smokers
with greater
oxidative stress,
additional vitamin C is needed
to restore
the α-tocopheroxyl
radical to its reduced
form. Importantly, vitamin C
supplementation
did not change F2-isoprostane
concentrations, showing that
α-tocopherol does
not prevent
radical formation or the
initial oxidation of fatty acids, but halts the
chain reaction of lipid peroxidation.
For further
information
Food and Nutrition Board, and
Institute of
Medicine.
Dietary reference
intakes for
vitamin C,
vitamin
E, selenium,
and carotenoids. Washington,
DC: National Academy
Press; 2000.
Institute
of Medicine.
Dietary reference
intakes: the
essential
guide to nutrient
requirements.
Washington, DC: National Academy
Press; 2006.
Manor D, Morley
S. The alpha-tocopherol
transfer protein.
Vitam Horm. 2007;76:45–65.
Traber
MG, Atkinson J. Vitamin
E, antioxidant and nothing
more. Free
Radic Biol Med. 2007;43:4–15.
Traber
MG, Stevens
JF. Beneficial
effects
from a mechanistic perspective.
Free Radic Biol Med.
2011;51:1000–13.
Traber
MG. Vitamin
E. In:
Erdman J, Macdonald I, Zeisel
S, editors. Present
knowledge
in nutrition. 9th ed.
Washington, DC: ILSI Press.
**************************************************************************
Four new quassinoids from the roots of Eurycoma longifolia Jack.
Fitoterapia. 2014 Jan;92:105-10
Authors: Meng D, Li X, Han L, Zhang
L, An W, Li X
Abstract
Seven compounds were isolated from the roots of Eurycoma longifolia, and
characterized by comprehensive analysis of 1D and 2D NMR experiments
along with single crystal X-ray diffraction. Among them, four new
quassinoids were identified and three of them
were diastereomers
for each other. Compounds
1-7 were evaluated for cytotoxicities against HT-29, MCF-7, LOVO,
BGC-823, MGC-803, HepG2, HeLa, and A549 cancer cell lines. Compounds 2
and 5 exhibited the lowest IC50 values
of
24.9 μM, 11.8 μM, and 44.1 μM, 14.1 μM towards MCF-7, MGC-803 cancer
cell lines, respectively, while compound 6 exhibited moderate
cytotoxicity towards all the selected cancer cell lines.--PMID: 24513570
[PubMed - indexed for MEDLINE]
********************************************************************
Anti-tumor activity of Eurycoma longifolia root extracts against K-562
cell line: in vitro and in
vivo study.
PLoS One. 2014;9(1):e83818
Authors: Al-Salahi OS, Ji D, Majid
AM, Kit-Lam C, Abdullah WZ, Zaki A, Jamal Din SK, Yusoff NM, Majid AS
Eurycoma longifolia Jack has been widely
used in traditional medicine for its antimalarial, aphrodisiac,
anti-diabetic, antimicrobial and anti-pyretic activities. Its anticancer
activity has also been recently reported on different solid tumors,
however no anti-leukemic activity of this plant has been reported. Thus
the present study assesses the in vitro and in vivo anti-proliferative
and apoptotic potentials of E. longifolia on K-562 leukemic cell line.
The K-562 cells (purchased from ATCC) were isolated from patients with
chronic myelocytic leukemia (CML) were treated with the various
fractions (TAF273, F3 and F4) of E.
longifolia root methanolic extract at various concentrations and time
intervals and the anti-proliferative activity assessed by MTS assay.
Flow cytometry was used to assess the apoptosis and cell cycle arrest.
Nude mice injected subcutaneously with 10(7) K-562 cells were used to
study the anti-leukemic activity of TAF273 in vivo. TAF273, F3 and F4
showed various degrees of growth inhibition with
IC50
values
of 19, 55 and 62 µg/ml, respectively. TAF273 induced apoptosis in a dose
and time dependent manner. TAF273 arrested
cell cycle at G1 and S phases. Intraperitoneal administration of TAF273
(50 mg/kg) resulted in a significant growth inhibition of subcutaneous
tumor in TAF273-treated mice compared with the control mice (P = 0.024).
TAF273 shows potent anti-proliferative activity in vitro and in vivo
models of CML and therefore, justifies further efforts to define more
clearly the potential benefits of using TAF273 as a novel therapeutic
strategy for CML management.--PMID:
24409284 [PubMed - indexed for MEDLINE]
*************************************************************************
Aspartame Toxicity Information
12 East Side Dr., Suite 2-18
Concord, NH 03301
603-225-2110
Date: January 12, 2002
Please find below Evidence File #4: Reported Aspartame Toxicity Effects
Reported Aspartame Toxicity Effects
-----------------------------------
Q. What are the reported reactions to aspartame ingestion?
How often are such effects seen?
Answer
------
==> What are the reported reactions to aspartame ingestion?
We will limit our discussion in this FAQ to reported toxicity
reactions to aspartame ingestion. Controlled studies showing
problems with aspartame ingestion will be discussed in another
FAQ. Toxicity reactions to aspartame can be divided into three
types:
1. Acute toxicity reactions occuring within 48 hours of ingestion of
an aspartame-containing product.
2. Chronic toxicity effects occuring anywhere from several days of
use to appearing a number of years (i.e., 1-20+ years) after the
beginning of aspartame use.
3. Potential toxicity effects that would be nearly impossible for
the user to recognize the link to aspartame.
In an epidemiological survey which appeared in the Journal of
Applied Nutrition (Roberts 1988), 551 persons who have
reported toxicity effects from aspartame ingestion were
surveyed. The adverse effects found cover a subset of reported
acute and chronic toxicity effects from aspartame.
What follows is a listing of the adverse health effects
which were found.
-------------------
# of
people (%)
Eye
- Decreased vision and/or other eye problems 140 (25%)
(blurring, "bright flashes," tunnel vision)
- Pain (or or both eyes) 51 (9%)
- Decreased tears, trouble with contact lens, 46 (8%)
or both
- Blindness (one or both eyes) 14 (3%)
Ear
- Tinnitus ("ringing," "buzzing") 73 (13%)
- Severe intolerance for noise 47 (9%)
- Marked impairment of hearing 25 (5%)
Neurologic
- Headaches 249 (45%)
- Dizziness, unsteadiness, or both 217 (39%)
- Confusion, memory loss, or both 157 (29%)
- Severe drowsiness and sleepiness 93 (17%)
- Paresthesias ("pins and needles," "tingling") 82 (15%)
or numbness of the limbs
- Convulsions (grand mal epileptic attacks) 80 (15%)
- Petit mal attacks and "absences" 18 (3%)
- Severe slurring of speech 64 (12%)
- Severe tremors 51 (9%)
- Severe "hyperactivity" and "restless legs" 43 (8%)
- Atypical facial pain 38 (7%)
Psychologic-Psychiatric
- Severe depression 139 (25%)
- "Extreme irritability" 125 (23%)
- "Severe anixiety attacks" 105 (19%)
- "Marked personality changes" 88 (16%)
- Recent "severe insomnia" 76 (14%)
- "Severe aggravation of phobias" 41 (7%)
Chest
- Palpitations, tachycardia (rapid heart action), 88 (16%)
of both
- "Shortness of breath" 54 (10%)
- Atypical chest pain 44 (8%)
- Recent hypertension (high blood pressure) 34 (6%)
Gastrointestinal
- Nausea 79 (14%)
- Diarrhea 70 (13%)
Associated gross blood in the stools (12)
- Abdominal pain 70 (13%)
- Pain on swallowing 28 (5%)
Skin and Allergies
- Severe itching without a rash 44 (8%)
- Severe lip and mouth reactions 29 (5%)
- Urticaria (hives) 25 (5%)
- Other eruptions 48 (9%)
- Aggravation of respiratory allergies 10 (2%)
Endocrine and Metabolic
- Problems with diabetes: loss of control; 60 (11%)
precipitation of clinical diabetes;
aggravation or simulation of diabetic
complications
- Menstrual changes 45 (6%)
Severe reduction or cessation of periods (22)
- Paradoxic weight gain 34 (5%)
- Marked weight loss 26 (6%)
- Marked thinning or loss of the hair 32 (6%)
- Aggravated hypoglycemia (low blood sugar 25 (5%)
attacks)
Other
- Frequency of voiding (day and night), burning 69 (13%)
on urination (dysuria), or both
- Excessive thirst 65 (12%)
- Severe joint pains 58 (11%)
- "Bloat" 57 (10%)
- Fluid retention and leg swelling 20 (4%)
- Increased susceptibility to infection 7 (1%)
-------------------
There are other clinical reports in the scientific literature of
aspartame-caused toxicity reactions including Blumenthal (1997),
Drake (1986), Johns (1986), Lipton (1989), McCauliffe (1991),
Novick (1985), Watts (1991), Walton (1986, 1988), and Wurtman
(1985).
Many pilots appear to be particularly susceptible to the effects of
aspartame ingestion. They have reported numerous serious toxicity
effects including grand mal seizures in the cockpit (Stoddard 1995).
Nearly 1,000 cases of pilot reactions have been reported to the
Aspartame Consumer Safety Network Pilot Hotline (Stoddard 1995).
This susceptibility may be related to ingesting methanol at altitude
as suggested in a letter from Dr. Phil Moskal, Professor of
Microbiology, Biochemistry, and Pathology, Chairman of the Department
of Pathology, Director of Public Health Laboratories (Moskal 1990),
or it may simply be that some pilots tend to ingest large quantities
of aspartame during a flight. Whatever the case, numerous warnings
about aspartame dangers have appeared in piloting journals including
The Aviation Consumer (1988), Aviation Medical Bulliten (1988),
Pacific Flyer (1988), CAA General Aviation (1989), Aviation Safety
Digest (1989), General Aviation News (1989), Plane & Pilot (1990),
Canadian General Aviation News (1990), National Business Aircraft
Association Digest (NBAA Digest 1993), International Council of
Air Shows (ICAS 1995), and the Pacific Flyer (1995). Both the U.S.
Air Force's magazine "Flying Safety" and the U.S. Navy's magazine,
"Navy Physiology" published articles warning about the many dangers
of aspartame including the cumlative deliterious effects of methanol
and the greater likelihood of birth defects. The articles note that
the ingestion of aspartame may make pilots more susceptible to
seizures and vertigo (US Air Force 1992).
Countless other toxicity effects have been reported to the FDA (DHHS
1995), other independent organizations (Mission Possible 1996,
Stoddard 1995), and independent scientists (e.g., 80 cases of
seizures were reported to Dr. Richard Wurtman, Food (1986)).
Samples of some aspartame toxicity reactions reported on the
Internet can be found on the Aspartame (NutraSweet) Toxicity Info
Center web page:
http://www.tiac.net/users/mgold/aspartame/
Frequently, aspartame toxicity is misdiagnosed as a specific disease.
This has yet to be reported in the scientific literature, yet it has
been reported countless times to independent organizations and
scientists (Mission Possible 1994, Stoddard 1995). In other cases,
it has been reported that chronic aspartame ingestion has triggered
or worsened certain chronic illnesses. Nearly 100% of the time, the
patient and physician assume that these worsening conditions are
simply a normal progression of the illness. Sometimes that may be
the case, but many times it is chronic aspartame poisoning.
According to researchers and physicians studying the adverse
effects of aspartame, the following list contains a selection
of chronic illnesses which may be caused or worsened by the chronic,
long-term ingestion of aspartame. (Mission Possible 1994, Stoddard
1995)*:
Brain tumors Multiple sclerosis
Epilepsy Chronic faigue syndrome
Parkinson's Disease Alzheimer's
Mental retardation Lymphoma
Birth defects Fibromyalgia
Diabetes Arthritis (including Rheumatoid)
Chemical Sensitivities Attention Deficit Disorder
*Note: In some cases such as MS, the severe symptoms mimic the illness or exacerbate the
illness, but do not cause the disease.
Also, please note that this is an incomplete list. Clearly,
ingestion of a very slow poison (as discussed in other FAQs) is not
beneficial to anyone who has a chronic illness.
Finally, potential toxicity effects from aspartame including brain
cancer (as seen in pre-approval research) and effects on fetal brain
and nervous system development will be discussed in other FAQs.
==> How often are such effects seen?
Until recently approximately 90% of aspartame sales were in the
United States (Monsanto 1994). Other countries are now being inundated
with aspartame, but it will be some time until they begin to feel the
full effects of aspartame toxicity on the general population. Since the
U.S. has some history of significant use, we will limit the discussion
to the frequency of effects in the U.S.
There have been well over 7,000 aspartame toxicity reactions officially received by the U.S.
Food and Drug Administration between 1982 (after aspartame was first approved) until 1995
(DHHS 1993, DHHS 1995). From this figure, we can estimate the number of actual toxicity
reactions observed.
***************************************************************************
References Cited
Aviation Consumer 1988. "SafeGuard," June 15, 1988.
Aviation Medical Bulletin 1988. "Pilots and Aspartame,"
October 1988.
Aviation Safety Digest 1989. "Aspartame -- not for the
dieting pilot?" Aviation Safety Digest, ASD 142, Spring
1989 (Australia - 062/5841111).
Blumenthal, H.J., D.A. Vance, 1997, "Chewing Gum Headaches,"
Headache, Volume 37, Number 10, pages 665-666.
Butchko, Harriett H., Frank N. Kotsonis 1994. "Postmarketing
Surveillance in the Food Industry: The Aspartame Case
Study," in Nutritional Toxicology, edited by Frank N.
Kotsonis, Maureen Macky and Jerry Hjelle, Raven Press,
Ltd., New York, c1994.
CAA General Aviation (1989). Safety Information Leaflet,
April 1989, Great Britain.
Canadian General Aviation News 1990. "Fit to fly" Canadian
General Aviation News, March 1990, page 28.
DHHS 1993. "Adverse Reactions Associated With Aspartame
Consumption," Department of Health & Human Services
Memorandum, April 1, 1993, Reprinted in preface of
"Bittersweet Aspartame: A Diet Delusion" by Barbara
Alexander Mullarkey, NutriVoice, P.O. Box 946, Oak
Park, Illinois 60303, (708) 848-0116.
DHHS 1995. Department of Health and Human Services. "Report
on All Adverse Reactions in the Adverse Reaction
Monitoring System." (April 20, 1995).
Drake, M.E., 1986. "Panic Attacks and Excessive Aspartame Ingestion"
(Letter), Lancet, September 13, 1986, page 631.
Food 1986. Food Chemical News, July 28, 1986, page 44.
Food 1995. "Aspartame Adverse Reaction Reports Down in 1994
From 1985 Peak: FDA," Food Chemical News, June 12, 1995,
page 27.
GAO 1986. "Six Former HHS Employees' Involvement in
Aspartame's Approval," United States General Accounting
Office, GAO/HRD-86-109BR, July 1986.
General Aviation News 1989. "NutraSweet...too good to be
true?" by Megan Hicks, General Aviation News, July 31,
1989.
Gordon, Gregory, 1987. "NutraSweet: Questions Swirl," UPI
Investigative Report, 10/12/87. Reprinted in US Senate
U.S. Senate Committee on Labor and Human Resources,
November 3, 1987 regarding "NutraSweet Health and Safety
Concerns." Document # Y 4.L 11/4:S.HR6.100, page 499.
ICAS 1995. "Aspartame Side Effects: Fact or Fiction?"
International Council of Air Shows, February 1995.
Johns, Donald R., 1986. "Migraine Provoked By Aspartame," (Letter),
New England Journal of Medicine, Volume 314, August 14, 1986,
page 456.
Kessler, David A. 1993, "Introducing MEDWatch: A New
Approach to Reporting Medication and Device Adverse
Effects and Product Problems" Journal of the American
Medical Association 269:2765-68.
Lipton, Richard B., et al., 1989. "Aspartame as a Dietary Trigger of
Headache," Headache, Volume 29, pages 90-92.
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**************************************************************************
Marianne Geiser,1
Barbara Rothen-Rutishauser,1
Nadine Kapp,1
Samuel Schürch,1,2
Wolfgang Kreyling,3
Holger Schulz,3
Manuela Semmler,3
Vinzenz Im Hof,4
Joachim Heyder,3 and
Peter Gehr1
Author
information ►
Article
notes ►
Copyright
and License information ►
See "Particles
in Practice: How Ultrafines Disseminate in the Body" on page A758a.
See letter "Translocation
of Ultrafine Particles" in volume 114 on page A211b.
This article has
been
cited by other articles in PMC.
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Abstract
High
concentrations of airborne particles have been associated with increased
pulmonary and cardiovascular mortality, with indications of a specific
toxicologic role for ultrafine particles (UFPs; particles < 0.1 μm).
Within hours after the respiratory system
is exposed to UFPs, the UFPs may appear in many compartments of the
body, including the liver, heart, and nervous system. To
date, the mechanisms by which UFPs penetrate boundary membranes and the
distribution of UFPs within tissue compartments of their primary and
secondary target organs are largely unknown. We combined different
experimental approaches to study the distribution of UFPs in lungs and
their uptake by cells. In the
in vivo experiments, rats
inhaled an ultrafine titanium dioxide aerosol of 22 nm count median
diameter. The intrapulmonary distribution of particles was analyzed 1 hr
or 24 hr after the end of exposure, using energy-filtering transmission
electron microscopy for elemental microanalysis of individual particles.
In an in vitro study,
we exposed pulmonary macrophages and red blood cells to fluorescent
polystyrene microspheres (1, 0.2, and 0.078 μm)
and assessed particle uptake by confocal
laser scanning microscopy. Inhaled ultrafine titanium dioxide particles
were found on the luminal side of airways and alveoli, in all major lung
tissue compartments and cells, and within capillaries.
Particle uptake in vitro
into cells did not occur by any of the expected endocytic processes,
but rather by diffusion or adhesive
interactions.
Particles within cells are not membrane bound
and hence
have direct access to intracellular proteins, organelles, and DNA, which
may greatly enhance their toxic potential.
Keywords:
aerosol, erythrocytes, lungs, macrophages, microscopy, nanoparticles,
rats, surfactant
High concentrations of airborne particles have been associated with
increased pulmonary and cardiovascular mortality, with indications of a
specific toxicologic role for ultrafine particles (UFPs; particles with
diameters < 0.1 μm) (Peters
et al. 1997). UFPs may induce
inflammatory and prothrombotic responses, promoting atherosclerosis,
thrombogenesis, and the occurrence of other cardiovascular events
(Schulz
et al. 2005). Human data suggest that
inhaled UFPs influence lung physiology (Pietropaoli
et al. 2004). UPFs may also affect the
autonomic nervous system or act directly on cells in various organs and
induce mutations (Harder
et al. 2005;
Samet et al. 2004). After exposure of the
respiratory system to UFPs, the UFPs may appear within hours in many
compartments of the body, including the liver, heart, and nervous system
(Brown
et al. 2002;
Kreyling et al. 2002;
Oberdörster et al. 2004).
UFPs are formed by
gas-to-particle conversion or by incomplete fuel combustion.
Despite considerable efforts to reduce air pollution, the
environmental burden by UFPs may have
increased rather than decreased over time (Kreyling
et al. 2003). Moreover, the
fast-growing nanotechnology industry generates new UFPs daily, which may
become aerosolized at some stage and may present additional health
risks. UFPs possess increased toxicity compared with larger particles
composed of the same materials
(Ferin
et al. 1992). Their environmental
burden is characterized by high number concentrations but low mass
concentrations. Thus, a relatively
large surface area per unit mass facilitates adsorption of various
organic compounds from the ambient air and enhances interaction with
biological molecules within the organism.
Deposition of UFPs
in the respiratory system is caused by diffusional displacement.
Depending on particle size, deposition occurs efficiently in the nose,
the conducting airways, and the alveoli. Although particles with
diameters > 1 μm usually remain on the epithelial surface upon their
deposition (Gehr
et al. 1990;
Geiser et al. 2003;
Schürch et al. 1990) and are subjected to
clearance by cough, mucociliary transport, and/or phagocytosis by
macrophages, UFPs seem to penetrate the boundary membranes of the lungs
rapidly—a unique feature for insoluble particles (Brown
et al. 2002;
Kreyling et al. 2002;
Oberdörster et al. 2002). In addition,
transport across the olfactory epithelium and accumulation in the brain
were reported for various UFP types (Oberdörster
2004).
In vitro experiments
revealed penetration of UFPs into mitochondria of macrophages and
epithelial cells that was associated with oxidative stress and
mitochondrial damage (Li
et al. 2003).
Because everyone on earth
inevitably inhales thousands to millions of UFPs with each breath, it is
important to assess health risks by UFP air pollution. The costs of
actions to be taken to reduce ambient
aerosol particles are high and will affect the economy greatly,
presenting an urgent need to clarify the fate of inhaled UFPs.
To date, the mechanisms by which UFPs
penetrate boundary membranes and the distribution of UFPs within tissue
compartments of their primary and secondary target organs are largely
unknown.---This study is the first to investigate the
distribution of inhaled UFPs within lungs
at the individual particle level and
combines different experimental approaches—an
in vivo inhalation study in rats and an in vitro cell
exposure study on pulmonary macrophages and red blood cells (RBCs).----In
the in vivo experiments, rats inhaled an ultrafine titanium
dioxide aerosol of 22 nm count median diameter (CMD) during 1 hr,
resulting in a deposition of 4–5 μg TiO2 per animal. The
intrapulmonary distribution of deposited particles was analyzed
immediately or 24 hr after the end of exposure, using energy-filtering
transmission electron microscopy (EFTEM) to allow elemental
microanalysis of individual particles (Kapp
et al. 2004).--In the in vitro study, we exposed cultured
porcine pulmonary macrophages and human RBCs to fluorescent polystyrene
microspheres with diameters of 1, 0.2, and 0.078 μm and assessed
particle uptake by confocal laser scanning microscopy (CLSM).
Materials and
Methods
Animals.
The animal
experiments were conducted under federal guidelines for the use and care
of laboratory animals (German Animal Protection Law) and were approved
by the District of Upper Bavaria (Approval No. 211-2531-108/99) and by
the GSF Institutional Animal Care and Use Committee, as well as in
accordance with the Swiss Federal Act on Animal Protection and the Swiss
Animal Protection Ordinance. Ten young, adult, male WKY/NCrl BR rats
[body weight (bw) 250 ± 10 g (mean ± SD; Charles River, Sulzfeld,
Germany] were housed under standard conditions, with access to food and
water ad libitum, in a room controlled for humidity (55%
relative humidity) and temperature (22°C) and with lighting on a 12-hr
day/night cycle. Animals were anesthetized by intramuscular injection of
a mixture of medetomidine (Domitor; 150 μg/100 g bw; Pfizer GmbH,
Karlsruhe, Germany), midazolam (Dormicum; 0.2 mg/100 g bw; Hoffmann-La
Roche AG, Grenzach-Wyhlen, Germany), and Fentanyl (0.5 μg/100 g bw;
Janssen-Cilag GmbH, Neuss, Germany) for inhalation and lung fixation and
anticoagulated by intra-peritoneal injection of 2,000 IU heparin
(Heparin-Natrium-ratiopharm, ratiopharm GmbH, Ulm/Donautal, Germany) for
lung fixation (Kapp
et al. 2004). Anesthesia was antagonized by subcutaneous injection
of atipamezole (Antisedan; Pfizer GmbH), flumazenil (Anexate,
Hoffmann-La Roche AG), and naloxone (Narcanti, Janssen Animal Health,
Neuss, Germany).
Aerosols and
inhalation.
Titanium is
suitable for EFTEM because it does not interfere with the heavy metals
used for tissue preparation (Kapp
et al. 2004). TiO2 is inert and
nonpathogenic (Templeton
1994), and ultrafine aerosols thereof remain as insoluble
particles (Donaldson
et al. 2002). Generally, commercially available TiO2
particles (Degussa, Düsseldorf, Germany) show a positive Zeta potential
of 20–30 mV as measured by Malvern Zetasizer 3000 HS (Malvern
Instruments Ltd., Malvern, Worcestershire, UK) The generation and
inhalation of the TiO2 aerosol used in this study has been
described in detail by
Kapp et al. (2004). Briefly, ultrafine TiO2 aerosols were
generated with a Palas spark generator (Palas GmbH, Karlsruhe, Germany)
in a pure argon plus 0.1% oxygen stream. The aerosol was
quasi-neutralized by a radioactive 85Kr source, diluted, and
conditioned for inhalation. Particle size distribution and number
concentration were monitored continuously by a differential electrical
mobility particle sizer and a condensation particle counter. An aerosol
of 22 nm CMD (geometric SD of 1.7) was produced. The mean number
concentration was 7.3 × 106 particles/cm3 (SD 0.5
× 106 particles/cm3), corresponding to a mass
concentration of 0.11 mg/m3. The 22 nm particles measured as
aerosol particles are already agglomerates of smaller primary structures
formed immediately after spark ignition and condensation.
The estimated size of the primary structures
was 4 nm as
derived from the measured specific surface area of 330 m2/g
of the UFPs produced in this study and the TiO2 bulk density
of 4.2 g/cm3.
Each anesthetized rat was placed in
an airtight plethysmograph box. The animals inhaled the aerosol in pairs
(one each for the 1-hr and 24-hr examinations) for 1 hr via an
endotracheal tube by negative-pressure ventilation (−1.5 Pa) at a
breathing frequency of 45/min, resulting in a minute volume of about 200
cm3/min (Kreyling
et al. 2002).
From breathing and aerosol parameters, the deposited amount of TiO2
was calculated to be 4–5 μg in each rat.
After aerosol exposure, anesthesia of one of the paired rats was
antagonized as described above, and the animal was returned to its cage
for 24 hr.
Lung fixation and
tissue preparation.
Lungs were fixed either
1 hr or 24 hr after the aerosol inhalation by sequential intravascular
perfusion with buffered 2.5% glutaraldehyde (Agar Scientific Ltd., Plano
GmbH, Wetzlar, Germany), 1.0% osmium tetroxide (Simec, Zofingen,
Switzerland), and 0.5% uranyl acetate
(Fluka Chemie GmbH, Sigma-Aldrich, Buchs, Switzerland). Lungs were then
subjected to systematic tissue sampling, dehydration in a graded series
of ethanol, and embedding in Epon (Fluka) (Im
Hof et al. 1989;
Kapp et al. 2004). Ultrathin (≤50 nm)
sections were cut from five to eight tissue blocks per animal, mounted
onto uncoated 600-mesh copper grids, and stained with uranyl acetate and
lead citrate (Ultrostain, Leica, Glattbrugg, Switzerland).
Particle
localization and elemental micro-analysis in situ.
On ultrathin
sections, 12 fields, corresponding to an area of 1,820 μm2
each, were systematically sub-sampled and investigated for the presence
and localization of TiO2 particles in a LEO 912 transmission
electron microscope (LEO, Oberkochen, Germany) equipped with an
in-column energy filter allowing energy dispersion for element specific
contrast. TiO2 particles were identified by parallel electron
energy-loss spectroscopy (parallel-EELS), electron spectroscopic
imaging, and image-EELS (Kapp
et al. 2004). For elemental micro-analysis, we used the L 2 , 3
edge of Ti at 456 eV energy loss. We obtained bright-field and
structure-sensitive micrographs (recorded at 250 eV), as well as
element-specific contrast for TiO2, by digital acquisition.
Cell culture
experiments.
Porcine lung
macrophages (provided by K. McCullough and H. Gerber, Institute for
Virus Diseases and Immune Prophylaxis, Mittelhäusern, Switzerland) were
cultured at 106 cells/mL in two-chamber slides (VWR
International AG, Dietikon, Switzerland) for 24 hr at 37°C and 5% CO2
in RPMI 1640 medium (containing 25 mM Hepes; LabForce AG, Nunningen,
Switzerland) with 10% fetal bovine serum (LabForce), 1%
l-glutamine (LabForce), and 1%
penicillin/streptomycin (Gibco BRL, Invitrogen AG, Basel, Switzerland).
Human RBCs, always freshly isolated from the same donor, were cultured
at 8 × 106 cells/mL and for 6–24 hr, as described above.
For CLSM, fluorescent polystyrene
microspheres with diameters of 1, 0.2, and 0.078 μm (Fluoresbrite plain
yellow green; Polysciences, Chemie Brunschwig AG, Basel, Switzerland) were added to
the cells at 1010 particles/mL in supplement-free RPMI 1640
medium for 4 hr. To inhibit phagocytic uptake, cells were pretreated
with cytochalasin D (cytD, 10 μg/mL; Fluka) for 30 min and during
incubation with particles (Thiele
et al. 2001). Macrophages were then
washed in phosphate-buffered saline (PBS), fixed in 3%
paraformaldehyde/PBS, treated with 0.1 M glycine/PBS, permeabilized with
0.2% Triton X-100/PBS, and stained for F-actin with rhodamine-phalloidin
(1:100; Molecular Probes, VWR International AG, Lucerne, Switzerland)
for 60 min at 37°C. RBCs were fixed in 2.5% glutaraldehyde/PBS,
resulting in a red autofluorescence. Preparations were mounted in
PBS:glycerol (2:1) containing 170 mg/mL Mowiol 4-88 (Calbiochem, VWR
International AG).
For TEM analysis,
RBCs were incubated with 0.025 μm gold particles (ANAWA Trading SA,
Wangen, Switzerland) at 6.6 × 1010
particles/mL. RBCs were fixed with buffered 2.5% glutaraldehyde, 1%
osmium tetroxide, and 0.5% uranyl acetate, and prepared for TEM as
described above for lung tissue.
Microscopic
analysis of cultured cells.
We used a
MicroRadiance system from Bio-Rad (Hemel Hempstead, UK) combined with an
inverted Nikon microscope (Eclipse TE3000; lasers: GHe/Ne 543 nm and Ar
488 nm) (Nikon, Küsnacht, Switzerland). Optical
sections with a voxel dimension of 50 × 50 × 200 nm were taken with a
100× /1.4 plan-apochromate objective. We performed image processing and
visualization using IMARIS software (Bitplane AG, Zurich, Switzerland).
We applied a deconvolution algorithm using Huygens2 software (Scientific
Volume Imaging B.V., Hilversum, Netherlands) to increase axial and
lateral resolutions and to decrease noise (Rothen-Rutishauser
et al. 1998). Experiments were performed in triplicate or
quadruplicate, and 30–50 cells were scanned by CLSM for each data point.
We examined ultrathin sections of RBCs incubated with gold particles in
a Philips 300 TEM at 60 kV (Philips, Zurich, Switzerland).
Go to:
Results
Distribution of inhaled UFPs in lungs.
We found TiO2
particles on the luminal side of airways and alveoli as well as within
each tissue compartment of the lung
(Figures
1 and
2).
Particles were localized within
epithelial and endothelial cells, within fibroblasts and between
collagen fibrils in the connective tissue, within blood capillaries, and
even within RBCs (Figure
1). Intracellular particles were localized most often in the
cytoplasm and rarely within the nucleus.
Particles within cells were not membrane bound. On average,
79.3 ± 7.6% (mean ± SD) of the particles were found on the luminal side
of airways and alveoli, 4.6 ± 2.5% were within epithelial or endothelial
cells, 4.8 ± 4.5% within the connective tissue, and 11.3 ± 3.9% within
the capillaries. The relative
distributions of particles among different lung compartments at 1 hr and
at 24 hr after inhalation were not different from each other and
correlated with the volume fractions of the respective compartments
(Figure
2).
Figure 1
EFTEM micrographs
of particles (arrows) in the lung parenchyma. Abbreviations: AL,
alveolar lumen; C, collagen fibril; CL, capillary lumen; EC,
erythrocyte; EN, capillary endothelial cell; EOS, eosinophil
granulocyte; EP, epithelium; F, fibroblast; FC, ...
Figure 2
Relative
distribution of particles localized in the different lung compartments
at 1 hr and 24 hr after inhalation. Volume densities (Vv) for
lung tissue compartments from
Burri et al. (1973),
Pinkerton et al. (1992), and
Tschanz et al. (1995,
2003).
Uptake of UFPs by
macrophages and RBCs.
We studied
the uptake of fine (1–0.2 μm) and ultrafine (< 0.1 μm) fluorescent
microspheres in cultured macrophages as a model for phagocytic cells and
in human RBCs as a model for nonphagocytic cells. Additionally, we
treated macrophage cultures with cytD to block phagocytosis. Cells were
prepared for CLSM, and a deconvolution algorithm was applied to increase
lateral as well as axial resolution. We found particles of all sizes
within macrophages (Figure
3A), however, with different percentages of cells involved in
particle uptake. On average, 77 ± 15% (mean ± SD) of the macrophages
contained UFPs, 21 ± 11% contained 0.2 μm particles, and 56 ± 30%
contained 1 μm particles (Figure
3B). CytD did not inhibit the uptake of ultrafine and 0.2 μm
particles but blocked the uptake of 1 μm particles by these cells (Figure
3B). We found ultrafine and 0.2 μm, but not 1 μm, particles in RBCs
(Figure
4A). TEM analysis of RBCs incubated with 0.025 μm gold particles
showed that intracellular particles were not membrane bound (Figure
4B).
Figure 3
(A) CLSM
micrographs of fluorescent polystyrene spheres (1, 0.2, and 0.078 μm)
taken up by macrophages in the absence (−) or presence (+) of cytD (bar
= 2 μm); F-actin is shown in red, and particles are green. The xy and xz
projections ...
Figure 4
(A) CLSM
micrographs of fluorescent polystyrene spheres taken up by RBCs.
Autofluorescence of the cells is shown in red, and particles are green
(bar = 2 μm). The xy and xz projections allow clear differentiation
between internalized (arrows) ...
Go to:
Discussion
The
ultrastructural analyses of lung tissue demonstrated that 1 hr after
aerosol inhalation, 24% of ultrafine TiO2 particles, on
average, were located within and beyond the epithelial barrier (i.e., in
the main lung tissue compartments, in the cytoplasm, and in the nucleus
of the cells). These
results confirm data from human studies, where the inhalation of
ultrafine carbon particles affected pulmonary diffusing capacity (Pietropaoli
et al. 2004), suggesting that particles
in the interstitium have physiologic effects.
This study also provides evidence
for some particle translocation into the micro-vasculature. In previous
studies on iridium particles, we found minute fractions of particles
translocated into secondary target organs (Kreyling
et al. 2002;
Semmler et al. 2004b). Different particle materials—iridium versus
TiO2—may have resulted in different particle translocation
patterns, and we do not know the exact composition and structure of the
ultrafine particle surface, which is likely to influence particle
translocation. The present study focuses on particle distribution within
the primary target organ, the lung, and the results do not determine
which fraction of particles may have escaped the lung micro-vasculature
to be systemically circulated.
Particles found
within cells were not membrane bound, indicating a nonendocytic uptake.
In addition, the overall distribution pattern of the particles in the
lungs (i.e., the percentages of particles in the different lung
compartments) at 1 hr and at 24 hr after particle inhalation was the
same and was correlated with the volume densities of the corresponding
lung compartments, implying that ultrafine TiO2 particles can
move between tissue compartments without restraint. Our results are in
contrast with those obtained by
Stearns et al. (2001), who studied the uptake of ultrafine TiO2
particles in vitro in the A549 epithelial cell line. They found
that membrane-bound vesicles contained mostly large aggregates of TiO2.
Sometimes vesicles with clusters consisting of as few as two to three
particle profiles were observed. In a pilot study in vitro with
porcine macrophages, we obtained similar results (data not shown).
Whether these clusters contained few particles only or were sectioned
through the top of larger ones is not known. However, because ultrafine
TiO2 aggregate very quickly in polar liquids such as cell
culture medium and because particle concentration was fairly high, as
seen from micrographs, it is very likely that particles aggregated
within the cell culture medium and that cells engulfed these large
clusters by an endocytic pathway. Particle agglomeration on the lung
epithelium during the 1-hr inhalation, however, is very unlikely because
of the size of the inner lung surface and the number of deposited UFPs.
The fact that 80% of the retained
TiO2 particles were still on the luminal side of the
epithelium even 24 hr after inhalation is surprising and in contrast to
previous studies on the lavageability of ultrafine iridium particles,
where only 20% of the particles could be lavaged
from the epithelial surfaces 24 hr after inhalation (Kreyling
et al. 2002). The low lavageability of ultrafine iridium particles
in contrast to high lavageability of 80% of 0.5–10-μm particles (Oberdörster
et al. 2002) was interpreted as either
higher adhesion of UFPs to epithelial membranes or epithelial uptake and
penetration of UFPs into the interstitium. The large fraction
of TiO2 particles on the luminal side of the epithelium in
the present study strongly supports higher
adhesion of UFPs to epithelial structures.
However, it must also be considered that
proteins are very likely to bind rapidly to the particles, which then
may affect the further metabolic fate of the particles in terms of their
adhesion, residence time on the epithelium or uptake, and even
penetration through the epithelium.
Along this line,
the difference of the two particle materials
and surfaces may have led to binding of those proteins,
which then may have
mediated major uptake and penetration of iridium particles into and
through the epithelium.
We already have first evidence that ultrafine
commercial TiO2 particles bind more readily to other proteins
in the lung-lining fluid than carbonaceous and amorphous silica
particles (Semmler
et al. 2004a).
Microscopic analyses of phagocytic
and nonphagocytic cells incubated with different particle types showed
that macrophages take up fine and ultrafine polystyrene microspheres and
that treatment with cytD inhibits the uptake of 1.0-μm particles, but
not uptake of the smaller particles, by these cells. Ultrafine
polystyrene and gold particles also entered RBCs and were not membrane
bound.
The mechanisms
of intracellular uptake of macromolecules, particles, and even cells are
subsumed as endocytosis.
Material to be ingested is progressively enclosed by the plasma
membrane, which eventually detaches to form an endocytic vesicle.
Phagocytosis and pinocytosis are distinguished by the size of endocytic
vesicles formed. Phagocytosis, a receptor-mediated, actin-based process,
is characteristic for neutrophils, macrophages, and dendritic cells. It
is the main mechanism for the clearance of insoluble 1- to 3-μm
particles from the alveoli. Pinocytosis involves the ingestion of fluid
and solutes via vesicles of about 100 nm in diameter. There are at least
four basic mechanisms, most of which can be demonstrated in lungs and
involve specific receptor–ligand
interactions:
a) macro-pinocytosis,
b) clathrin-mediated, actin-based endocytosis, c)
caveolae-mediated endo- or transcytosis, and d) clathrin- and
caveolae-independent endocytosis (Conner
and Schmid 2003).
Kruth et al. (1999) described an additional endocytic process,
patocytosis, in which hydrophobic
polystyrene particles < 0.5 μm are transported through induced plasma
membrane channels into an extensive labyrinth of interconnected
membrane-bound compartments. None of these endocytic
pathways, all of which include vesicle formation, is likely to account
for the translocation of UFPs in our study, as intracellularly localized
particles were not membrane bound. Moreover, because RBCs contained UFPs
and cytD treatment of macrophages did not prevent UFP translocation into
these cells, particle uptake by any actin-based mechanism can also be
excluded.
Transport via pores,
as suggested for lung–blood substance exchange (Conhaim
et al. 1988;
Hermans and Bernard 1999), is another
potential mechanism for UFP translocation. TiO2 particles may
diffuse through such pores.
A transport mechanism by diffusion is consistent
with the observed spatial distribution of
UFPs in our inhalation study. Thus far, signal-mediated
transport via pores has been demonstrated only for
ultrafine gold particles of up to 39 nm in
diameter, through the nuclear pore complex in Xenopus oocytes,
where transport velocities depended on particle size (Panté
and Kann 2002).
Passive uptake (not triggered by
receptor–ligand interactions) may also occur
by electrostatic, Van der Waals, or steric interactions,
subsumed under “adhesive interactions” (Rimai
et al. 2000). Rimai et al. showed that 8-μm glass particles were
approximately 90% engulfed by a polystyrene substrate, compared with
22-μm particles, which were only 30% engulfed. However, the influence of
particle size on their engulfment was not clarified in these
publications.
Several concepts for
the nonspecific engulfment of particles through interfacial structures
(including cell membranes) have been suggested.
A thermodynamic model using the “wettability criterion” was successful
in predicting passive particle uptake, although it did not take into
account the elastic properties of the cell membrane (Chen
et al. 1997). In another thermodynamic analysis combined with a
molecular dynamics simulation,
Bresme and Quirke (1999) showed that line tension influences the
wetting behavior of nanoparticles at liquid–vapor and liquid–liquid
interfaces. These authors found negative line tension values for
particles of a few nanometers in diameter, but positive values for those
an order of magnitude larger. A negative line tension favors the initial
wetting of a spherical particle after its approach to an interface.
Shanahan (1990) studied the
engulfment of solid-surface heterogeneities equivalent to particles in
the nanometer range by unbalanced capillary forces (free energy
perturbations). Thermal
capillary waves cause fluid droplets to coalesce with a fluid substrate
by film drainage at the interface, breakage of the film, and intrusion
of the particle into the bulk phase (Aarts
et al. 2004). Thus, thermal capillary
fluctuations may enhance particle transport through cell membranes.
Experimental results demonstrate
consistently greater immersion of smaller particles than larger ones
into a liquid substrate covered by surfactant film in vitro as
well as in situ in airways. These results support the concept
that line tension plays a significant role in particle displacement (Geiser
et al. 2000;
Schürch et al. 1999).
It remains to be determined which
chemical and physical properties of membranes and particles are
responsible for the translocation of UFPs in vivo.
Interestingly, we did not see any difference in particle uptake in
vitro with respect to differing surface charges or surface
chemistry when we used three different particle types: a metal, a metal
oxide, and a synthetic polymeric material (data not shown). However,
these particles were added to the cells in suspension and did not
approach the cells from the air or require
passage through a surfactant film first, as in the in vivo
inhalation experiments. In these
in vivo experiments, electrostatic interactions are likely
important for particle deposition and subsequent retention.
In summary, UFPs of
various materials can cross any cellular membrane, but neither
endocytosis, which is based on vesicle formation, nor any actin-based
mechanisms are likely to account for UFP translocation into the cell.
Our results from the inhalation experiments with TiO2
particles point to a transport mechanism that includes adhesive
interactions or, in terms of thermodynamics, interfacial and line
tension effects. In
addition, particle diffusion and uptake promoted by thermal capillary
waves might play a role in particle transport through membranes.
After the deposition of nanometer-size
particles, their further fate may be largely independent from particle
surface chemistry and charge.
Consequently, it is
a possible fate of inhaled ambient UFPs that they are transferred from
the lungs to most other organs.
In a first analysis of ultrathin sections
from hearts of the same rats, we found ultrafine TiO2
particles in the connective tissue, that is, within fibroblasts. There
may be no means on the cellular level to prevent, influence, or direct
their uptake. Moreover, the
toxic potential of UFPs is greatly enhanced by their free location and
movement within cells, which promote interactions with intracellular
proteins and organelles and even the nuclear
DNA.
Potential health implications of our findings are related not only to
ambient UFPs but also to engineered “nanoscaled particles,” which may be
released into our environment during their production, transport, and
aging, or during waste disposal
(The
Royal Society 2004). Routes of
exposure to nanomaterials include oral, cutaneous, and inhalative
uptake, the latter being addressed in the present study. Although the
number of particles translocated into the cells may vary substantially
(Nanosafe 2004) according to their physicochemical properties, the data
of the present study strongly suggest that adverse health outcomes
associated with the uncontrolled presence of nanoscale particles in
tissues require further attention.
Footnotes
We thank S.
Frank, B. Haenni, B. Kupferschmid, and B. Tschirren for excellent
technical assistance and L.M. Cruz-Orive for his help with the lung
sampling design.
This study
was supported by the Swiss National Science Foundation; the Swiss Agency
for the Environment, Forest, and Landscape; and the Silva Casa
Foundation.
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TOP B
This
is the medical side but in the alternative 200IU to 400 IU of
Vitamin E was base----
To convert IUs of vitamin E into milligrams of alpha-tocopherol:
multiply the number of IUs by 0.67.
To convert milligrams of alpha-tocopherol to IUs: multiply the
number of milligrams by 1.5.
So 200IU X 0.67 = 13.4 mgs
400 IU X 0.67 = 26.8 ,gs
IC50 is the acronym for “half maximal
inhibitory concentration”. IC50 value indicates the
concentration needed to inhibit a biological or biochemical
function by half (e.g. inhibition of enzymes, affinity to cell
receptors). In pharmaceutical research, it is a frequently used
unit to specify the in vitro potency of a drug or a NCE.
Amongst others, determination of IC50 is commonly
calculated via linear interpolation: The activity of an enzyme is
determined after exposure to a series of inhibitor concentrations.
IC50 is calculated by the following formula:
IC50 = (50%-LowInh%)/(HighInh%-LowInh%)x(HighConc-LowConc)
+ LowConc
LowInh% / HighInh%: % inhibition directly
below / above 50% inhibition
LowConc / HighConc: Corresponding
concentrations of test compound
IC50 is the acronym for “half maximal inhibitory
concentration”. IC50 value indicates the concentration
needed to inhibit a biological or biochemical function by half
(e.g. inhibition of enzymes, affinity to cell receptors). In
pharmaceutical research, it is a frequently used unit to specify
the in vitro potency of a drug or a NCE.
Amongst others,
determination of IC50 is commonly calculated via linear
interpolation: The activity of an enzyme is determined after
exposure to a series of inhibitor concentrations. IC50
is calculated by the following formula:
IC50 = (50%-LowInh%)/(HighInh%-LowInh%)x(HighConc-LowConc)
+ LowConc
LowInh% / HighInh%: % inhibition directly
below / above 50% inhibition
LowConc /
HighConc: Corresponding concentrations of test compound
