A cross-sectional study to assess night-shift work related exposures and their association with markers of biological perturbation

Night-shift work impact

Night work has become an unavoidable part of our industrial society. In
developed countries approximately 15-20% of the working population is working
in shifts and this percentage is increasing. There is growing evidence that
shift work, and especially night-shift work, disrupts lifestyle and circadian
rhythm and therefore contributes to the development of chronic disorders,
including cardio-metabolic diseases and cancer. In 2007, the IARC
(International Agency for Research on Cancer) classified shift work involving
circadian disruption as probably carcinogen in humans.

Previous research

In the last decades many studies have assessed the effects of night-shift work
on health-related outcomes, with inconsistent results. In most cohort studies
that have been conducted to date night work is assessed using simple metrics
(e.g. *ever done night work* , or *duration of years of night work* ). Also,
previous research has been performed on effects of night-work on intermediate
markers, including hormonal levels, clock gene expression, and alertness. So
far, not much research has been performed on long-term or chronic disruptions
in response to night work between recently started and more experienced night
workers. Effects of night work on reproductive hormones and clock gene
expression is still a matter of debate since findings vary between previously
conducted studies.

Research gap

Even though the term night-shift work encompasses a wide range of different
*exposure* aspects, night-shift work is in previous studies often not further
defined, leading to a knowledge gap in actual mechanisms through which
night-shift work might have an impact on health. To better assess the potential
causal link between shift work and chronic disorders, we will need to
appreciate details of night work. The following night-shift work aspects
(potential mechanisms) have been hypothesized: light at night, nutrition at
night, disturbed sleep pattern, disturbed social pattern, altered amount of sun
exposure, and behavioral changes (diet, physical activity). Hypothesis on
mechanisms relating night-shift work to health are mainly generated in animal
research and it is often unclear how to translate these findings to human
studies. More information on these mechanisms, and more specific methods to
measure shift-work aspects in studies researching the effects of shift work,
would likely increase the statistical power to identify associated health
effects, and would provide a possible solution for inconsistency in results of
shift work research.

Another important research gap is the lack of long-term circadian disruption
biomarkers. Chronic circadian disruption is considered an important mechanism
between night-shift work and adverse health effects, yet, no biomarkers are
available to assess long-term or chronic circadian disruption. Long-term
markers would be useful for cohort studies and would possibly identify high
risk groups or behaviors related to night work. Moreover, when researching both
night-shift work aspects and biomarkers, biomarkers can be related to specific
aspects of night work instead of night work in general, resulting in more
informative conclusions about which aspects are most disruptive (reflect most
disruptions in biomarkers). We will use OMICs techniques (i.e. transcriptomics,
epigenetics, proteomics) to agnostically investigate perturbations on a
molecular level to generate new hypotheses for future research. Animal studies
using OMICs technologies have already identified potential long-term/chronic
disruption biomarkers. These results provide support for using these
technologies in identifying chronic disruption biomarkers.

This study aims at, first, assessing the most relevant exposure aspects of
night-shift work and, second, identifying biomarkers for both acute and chronic
circadian disruption associated to these specific nightshift work aspects. By
applying detailed questionnaires and objective monitors we will be able to
quantify specific aspects of the complex mixture of exposures together defined
as *night-shift work*. We will associate these aspects with intermediate health
outcomes, including known biomarkers of circadian disruption, to identifying
which aspects contribute most to biological perturbations. In addition, we aim
to identify new biomarkers reflecting chronic circadian disruption due to
night-shift work.

In the sub-study:

The goal of the Klokwerk-licht study is to identify new biomarkers reflecting
chronic circadian disruption due to night-shift work.

The design of the study will be cross-sectional. We will include 300 female
nurses. The participants will be divided into three groups: recently started
night-shift workers (<2 year), long-term night-shift workers (working in night
shifts for over 5 years) and non-shift workers (controls* did not perform night
or shift work >10 years). Night-shift workers and controls will be included in
our study for six (2x two consecutive days and 1x two consecutive nights) and
four (2 x two consecutive days) days, respectively. Exposure measurements will
be conducted continuously for the duration of the study, while biological
sampling will be conducted at the end of each two day (night) session.

After inclusion in the study, participants will receive a baseline
questionnaire via regular mail. Via the questionnaire we will assess baseline
characteristics of the study participants and previous exposure to night-shift
work. At the beginning of the study we will distribute an actimetry device and
a light sensor. We will ask participants to keep a short daily log, including
questions regarding sleep behavior, work (shift) schedule, activity and other
health behavior for the full duration of the study.

In addition, on the second day of each two-day session we will ask participants
to keep a log regarding their dietary pattern as well. We will distribute a
smart phone (used to conduct a psychomotor vigilance task (PVT) test). On the
second day of each two-day session biological sampling will take place.
Biological sampling will consist of whole blood, 24hour urine, hair and a feces
spot sample.

Collection of biological samples for a day-shift session will take place during
an afternoon shift, because during this shift participants are least disrupted.
Biomarker sampling after a night shift will take place at 8.00 AM, directly
after the night shift. Participants will fill out a biological questionnaire
for each two-day session to assess recent disease and medication use among
other parameters that might potentially disturb biomarker measurements.


In the sub-study:
The original Klokwerk study (running until the end of 2016) is a
cross-sectional study conducted among 150 nurses. Within this study population
an intensive study protocol (taking a maximum of 6 days to complete) has been
applied. The protocol consists of filling out questionnaires, wearing sensors,
and donating biological material. Within the Klokwerklicht study a small part
of the protocol will be applied among a group of 150 individuals that have not
yet participated in the original Klowkerk study. The protocol consists of
filling out two questionnaires and donating blood. We will use the same study
groups as in the original Klokwerk study: nurses/paramedics that never worked
in night shifts, nurses/paramedics that worked less than 2 years in night
shifts, and nurses that worked more than 5 years in night shifts. Participants
will be recruited from the hospital and healthcare centers where the original
Klokwerk study is conducted.

As this is an observational study the risk of adverse events due to
participation in this study is negligible. Burden for study participants
involves filling out questionnaires and logs, conducting Psychomotor Vigilance
Task tests, wearing light and activity measuring devices for the duration of
the study, and biological sampling. Slight inconvenience for the subjects is
expected resulting from wearing devices. Subjects will wear an actigraphy
device, day and night for two days in a row (four days in total for control
subjects and six days in total for night workers), in order to measure sleep,
movement and activity levels. Furthermore, at the same time, they will wear a
UV-sensitive light sensor to record UV and light intensity received by a
participant. At baseline the participants have to fill in an extensive
questionnaire and during the measurement period of two times two days they will
also keep a short daily log about their work schedule, sleep and health
behavior. On the days we collect blood samples (two times in total for control
subjects and three times in total for night workers), we will ask them to fill
out a nutritional log as well. This might take some time during work and might
therefore be inconvenient as well. Finally, 24-hr urine, feces, hair, and a
blood sample (4 tubes, 21 ml in total) will be collected from the subject.
Subjects might experience discomfort due to the collection of biological
samples. Collecting blood may provoke a hematoma which usually disappears fast.
Also the collection of hair might cause reluctant reactions, though this does
not lead to any health risk for the participant. Collecting feces and 24-hour
urine might make participants uncomfortable. In order to make the study more
feasible, the biomarkers are divided in primary and secondary biomarkers.
Primary biomarkers are of high priority, and crucial to answering the main
question of the study. Willingness to participate in these measurements are
part of the inclusion criteria of the study. Secondary biomarkers are needed to
answer additional research questions and of less priority. In total the contact
duration between the researchers and the participants regarding the entire
study will be approximately 1.5 hours.

The participants have no direct benefit from participating in the CLOCK WORK
study. This research can provide useful information for society. It can give a
better understanding of the potential adverse effects of night work on humans.
Desired, participants will receive a personal report with the most important
and interpretable outcome measures. The report will provide data on
chronobiology (day / night rhythm), blood pressure, cholesterol, glucose, sleep
outcomes, BMI, and complete blood counts. DNA measurements in blood are not in
this report, since the measured values only have meaning at group level.
However, after the entire research project is finished, the participants will
receive a report of the main results of the study. If test results from this
study suggest a health hazard for the participant, we will report to the
participant (if desired). If
the participant wishes, we will also tell her doctor about this test results.